U.S. patent application number 11/494458 was filed with the patent office on 2006-12-14 for icbp90 polypeptide and its fragments and polynucleotides coding for said polypeptides and applications for diagnosing and treating cancer.
This patent application is currently assigned to Institut National de la Sante et de la Recherche Medicale. Invention is credited to Christian Bronner, Raphael Hopfner, Jean-Marc Jeltsch, Yves Lutz, Marc Mousli, Pierre Oudet.
Application Number | 20060281125 11/494458 |
Document ID | / |
Family ID | 9547143 |
Filed Date | 2006-12-14 |
United States Patent
Application |
20060281125 |
Kind Code |
A1 |
Bronner; Christian ; et
al. |
December 14, 2006 |
ICBP90 polypeptide and its fragments and polynucleotides coding for
said polypeptides and applications for diagnosing and treating
cancer
Abstract
The invention concerns a novel ICBP90 (Inverted CCAAT box
binding protein 90) and its fragments, polynucleotides coding for
said polypeptides and specific antibodies directed against said
polypeptides. The invention also concerns methods and kits for
diagnosing cell proliferation and compounds useful as medicine for
preventing and/or treating pathology involving cell proliferation
and in particular cancer.
Inventors: |
Bronner; Christian;
(Fegersheim, FR) ; Hopfner; Raphael; (Strasbourg,
FR) ; Mousli; Marc; (Illkirch, FR) ; Jeltsch;
Jean-Marc; (Molsheim, FR) ; Lutz; Yves;
(Strasbourg, FR) ; Oudet; Pierre; (Strasbourg,
FR) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Institut National de la Sante et de
la Recherche Medicale
|
Family ID: |
9547143 |
Appl. No.: |
11/494458 |
Filed: |
July 28, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10019071 |
May 15, 2002 |
|
|
|
PCT/FR00/01747 |
Jun 22, 2000 |
|
|
|
11494458 |
Jul 28, 2006 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/320.1; 435/325; 435/69.1; 435/7.23; 530/350; 530/388.8;
536/23.5 |
Current CPC
Class: |
A61K 47/6843 20170801;
Y10S 530/827 20130101; Y10S 530/828 20130101; A61K 38/00 20130101;
A61P 35/00 20180101; C12Q 2600/136 20130101; C12Q 1/6886 20130101;
C07K 16/18 20130101; A61K 48/00 20130101 |
Class at
Publication: |
435/006 ;
435/007.23; 435/069.1; 435/320.1; 435/325; 530/350; 530/388.8;
536/023.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/574 20060101 G01N033/574; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C07K 14/82 20060101
C07K014/82; C07K 16/30 20060101 C07K016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 22, 1999 |
FR |
99 07935 |
Claims
1-17. (canceled)
18. A monoclonal or polyclonal antibody and its fragments, which
specifically binds a first polypeptide that a) comprises the amino
acid sequence SEQ ID NO. 2 or b) consists of a fragment of the
amino acid sequence SEQ ID NO. 2, wherein said fragment comprises
amino acids 263-793 of the amino acid sequence SEQ ID NO. 2.
19. The monoclonal antibody according to claim 18, wherein said
antibody is specific for the first polypeptide and is capable of
inhibiting the interaction between the first polypeptide and a DNA
sequence, to which the first polypeptide is specifically bound.
20. The monoclonal antibody according to claim 18, wherein said
antibody is specific for the first polypeptide and is capable of
inhibiting an interaction between the first polypeptide and a
second polypeptide, said second polypeptide i) comprises the amino
acid sequence SEQ ID NO. 2 or ii) is a protein of a transcriptional
complex.
21. A method for detecting and/or measuring a polypeptide, that a)
comprises the amino acid sequence SEQ ID NO. 2 or b) consists of a
fragment of the amino acid sequence SEQ ID NO. 2, wherein said
fragment comprises amino acids 263-793 of the amino acid sequence
SEQ ID NO. 2, in a biological samplecomprising: a) contacting the
biological sample with the monoclonal or polyclonal antibody of
claim 18; and b) revealing a formed antigen-antibody complex.
22. A kit for detecting and/or measuring a polypeptide that a)
comprises the amino acid sequence SEQ ID NO. 2 or b) consists of a
fragment of the amino acid sequence SEQ ID NO. 2, wherein said
fragment comprises amino acids 263-793 of the amino acid sequence
SEQ ID NO. 2, in a biological sample by immunological reaction,
comprising: a) the antibody of claim 18; b) if applicable, medium
reagents for formation of the favorable medium for the
immunological reaction; and c) detection reagents enabling
detection of the antigen-antibody complex produced by the
immunological reaction.
23-37. (canceled)
38. A composition for detecting, localizing and imaging cancers,
comprising the antibody according to claim 18, wherein the antibody
is labeled directly or indirectly with a marker generating a
signal, said marker is selected from the group consisting of
radioactive isotopes and nonisotope entities.
39. A method for detecting, localizing and imaging cancer cells,
comprising: a) injecting parenterally the composition according to
claim 38 in a human being; b) penetrating the labeled antibody
within said cancer cells, without said antibody being bound
substantially to normal cells; c) detecting a signal from the
labeled antibody by means of a signal doctor; and d) converting the
detected signal into an image of cancer cells.
40. A method for detecting and/or measuring a polypeptide that a)
comprises the amino acid sequence SEQ ID NO. 2 or b) consists of a
fragment of the amino acid sequence SEQ ID NO. 2, wherein said
fragment comprises amino acid 263-793 of the amino acid sequence
SEQ ID NO. 2, a biological sample, comprising: a) contacting the
biological sample with an antibody, which is specfic to the
polypeptide and is capable of inhibiting an interaction between the
polypeptide and a DNA sequence, to which the polypeptide is
specifically bound; b) and revealing a formed antigen-antibody
complex.
41. A method for detecting and/or measuring a polypeptide according
that a) comprises the amino acid sequence SEQ ID NO. 2 or b)
consists of a fragment of the amino acid sequence SEQ ID NO. 2,
wherein said fragment comprises amino acids 263-793 of the amino
acid sequence SEQ ID NO. 2, in a biological sample, comprising: a)
contacting the biological sample with an antibody, which is
specific to the polypeptide and is capable of inhibiting an
interaction between the polypeptide and a protein, with which the
polypeptide interacts, said protein i) comprises the amino acid
sequence SEQ ID NO. 2 or ii) is a protein of a transcriptional
complex; b) and revealing a formed antigen-antibody complex.
Description
[0001] This application is a divisional of application Ser. No.
10/019,071, which is a National Stage application of
PCT/FR00/01747, filed Jun. 22, 2000, which claims priority from
French patent application FR 99 07935, filed Jun. 22, 1999. The
entire contents of each of the aforementioned applications are
incorporated herein by reference.
[0002] The present invention relates to a new ICBP90 polypeptide
and its fragments, to the cloning of cDNA and polynucleotides
coding for said polypeptides, to cloning and/or expression vectors
including said polynucleotides, cells transformed by said vectors
and specific antibodies directed against said polypeptides. The
invention also relates to methods and kits for diagnosing cancers,
to a method and kit for screening ligands of the polypeptides of
the invention and of compounds which may be used as a drug for
prevention and/or treatment of cancers.
[0003] DNA topoisomerases are highly preserved nuclear proteins
during evolution, the main role of which is for controlling DNA
conformation and topology in the nucleus, which are constantly
altered by the various biological processes involving DNA such as
for example, transcription and replication. Topoisomerases exert
their action by cutting DNA and linking these lesions after having
achieved the adequate conformational change.
[0004] In mammals and humans in particular, today, there are at
least five different genes coding for a topoisomerase and at least
two additional pseudogenes (for a review, see Nitiss 1998). Thus,
topoisomerase I, coded by the TOP1 gene removes the superturns
present in DNA while only cutting a single strand. Both
topoisomerases of type II existing in humans called TopII.alpha.
and TopII.beta., alter DNA topology by introducing transient double
strand cleavages (for a review, see Wang 1996). Finally, there are
two topoisomerases of type III coded by two localized genes in
17p11.2-12 and 22q11-12 and they only act against negative
superturns of DNA.
[0005] In tumoral cells, topoisomerases of type II play a very
important role; in these growing and rapidly dividing cells, there
is a large need for maintaining DNA molecules in a proper
conformation as high transcription and replication rates are
required. Thus, the rates for topoisomerase II are generally higher
in human tumoral cells than in normal tissues of the same origin.
However, the high expression rate of topoisomerase II.alpha. in
tumoral cells may vary among two tumors of different natures
affecting a same tissue. For example, the nucleus of cells from
small cell carcinomas of the lung has a higher rate of
topoisomerase II.beta. than the nucleus of cells from lung
carcinomas with normal sized cells (Guinee et al., 1996). In the
same way, the rate of topoisomerase II.alpha. in A59 cells is three
times higher than in PC3 cells, both of these cell lines stemming
from the adenocarcinoma of lung epithelium (Yamasaki et al.,
1996).
[0006] These observations suggest that topoisomerase IIa may be
considered as a marker of cell proliferation for certain types of
cancer. As the cancerous process is characterized by abnormal cell
proliferation partly due to the loss of contact inhibition,
topoisomerase IIa therefore appears as a preferential target for
chemiotherapeutical drugs for treating cancer (Pommier et al.,
1994), and the present anticancer treatments largely resort to
inhibitors of topoisomerases.
[0007] Most of these inhibitors exert their cytotoxic effects by
stabilizing the DNA cleavage complex. Drugs like anthracyclines
[doxorubicin (adriamycin) or epipodophyllotoxins (such as etoposide
(VP-16) or teniposide (VM26))], acridines (such as mAMSA) and
anthracendiones (for example, mitoxantrone) are examples of drugs
which inhibit topoisomerases II which stabilize the cleavage
complex. More recently, a new class of inhibitors of topoisomerases
II has been developed; these inhibitors act at the level of
catalytic activity and no longer by stabilizing the cleavage
complex. The drug, fostriecin is an example of one of them
(Boritzki et al., 1988). Today these different drugs are used in
palliative and curative anticancer treatments.
[0008] Nevertheless, one of the major problems encountered in the
present anticancer treatments using inhibitors of topoisomerases is
the emergence of a resistance to drugs (Kubo et al., 1995). These
resistances are either the occurrence of an overexpression of pumps
providing efflux of drugs outside the cells before they reach their
target (for example; P-glycoprotein, a protein associated with
multi-drug resistance (MRP)), or the occurrence of a change in the
expression rate of topoisomerase II.alpha. (Deffie et al., 1989;
Fry et al., 1991), or either both occurrences (for a review, see
Isaacs et al., 1998).
[0009] One of the aspects of the present invention is therefore to
understand the regulatory mechanisms of the expression of the gene
of topoisomerase II.alpha., in order to develop an alternative to
the phenomenon of resistance to drugs, observed for certain cancers
and this with the aim of enhancing the curative and preventive
treatment of cancers.
[0010] There are two types of type II topoisomerase which differ in
their expression profile; topoisomerase II.alpha. (Top II.alpha.)
(170 kD), essentially located in the nucleoplasm at the centromer
of the mitotic chromosomes, participates in the fundamental
biological processes which are replication, condensation of
chromosomes and transcription. It seems that topoisomerase II.beta.
(Top II (180 kD) is rather involved in the transcription of
ribosomal RNA, given the nucleolar localization of this enzyme.
Both human type II topoisomerases are localized on two different
chromosomes (17q21-22 for topoisomerase II.alpha. and 3p24 for
topoisomerase II.beta.) (Tsai-Plugfelder et al, 1988; Drake et al.,
1989; Chung et al., 1989; Jenkins et al., 1992; Austin et al.,
1993).
[0011] Unlike topoisomerase II.beta., the expression of which is
characterized by a relative consistency, topoisomerase II.alpha.
has a variation of expression depending on the proliferation state
of cells and on their position in the cell cycle. Expression of
messenger RNA (RNAm) is higher in proliferating cells than in
arrested cells in confluence. The expression of topoisomerase
II.alpha. increases during the S phase of the cell cycle, reaching
a maximum at the end of phase G2/M (Goswami et al., 1996), the
level of messenger RNA being ten times higher at the end of phase S
than during phase Gi. Also, there seems to be a coupling between
the synthesis and degradation of topoisomerase II.alpha. and
chromosomal condensation/decondensation(Heck et al., 1988).
[0012] Present knowledge concerning control of the gene of
topoisomerase II.alpha., all in all, remains rather scanty.
Recently, a promoter region of about 650 base pairs has been
described by Hockhauser et al. (1992), it has all the
characteristics of a domestic gene, an absence of TATA box and a
moderate content of GC sites (notably the presence of a Sp1 box
which may replace the TATA box) are two examples of this. The
presence of inverted CCAAT boxes or ICBs is another feature of this
type of promoter.
[0013] Transcription factors interacting with the promoter of the
gene of human topoisomerase II.alpha. have been described; c-myb
(Brandt et al., 1997), p53 (Sandri et al., 1996), ATF (Lim et al.,
1998), Sp1 and Sp3 (Kubo et al., 1995) may be mentioned. Whatever
the case, apart from NF-Y (also called CBF, ACF and CP1, references
in Isaacs et al., 1996), the transcription factors which act on the
ICB sequences of the promoter for the gene of human topoisomerase
II.alpha. have not yet been identified and characterized; Herzog
and Zwelling (1997) have however revealed two proteins with an
apparent molecular weight of 90 kD and 140 kD which bind ICB1 to
ICB4 and ICB5, respectively. Isaacs and his collaborators (1996)
have suggested that NFY as well as another unidentified protein
recognize an ICB box of the promoter region of the gene of
topoisomerase II.alpha.; they have also shown that ICB2 mutations
completely suppressed the reduction in promoter activity normally
observed in cells arrested in confluence (Isaac et al., 1996). They
identified NFY as a component of a complex induced by the
proliferation and which binds in vitro to the ICN2 sequence of the
promoter of the gene of human topoisomerase II.alpha., although
NF-Y is always detectable in cells arrested in confluence (Isaacs
et al., 1996). They suggested that ICB2 acts as a negative
regulator of the promoter of the gene of topoisomerase II.alpha. of
cells arrested in confluence and that this repression may be
suppressed in proliferative cells. The ICB2 box of the promoter of
the gene of topoisomerase II.alpha. therefore plays a primordial
role in the arrest of the normal proliferative process when the
cells reach confluence.
[0014] Transcription factors binding to the ICB sequence as well as
the ICB sequence itself therefore form molecular targets for
controlling the expression rate of topoisomerase II.alpha.. By
intervening on these factors, controlling the expression of the
gene of topoisomerase II.alpha. and cell proliferation consequently
may be contemplated.
[0015] The object of the present invention is to detect new
transcription factors binding to the ICB box involved in the
control of cell proliferation.
[0016] A recent technique called a "simple hybrid" system has been
used, which allows DNAc clones coding for the proteins binding to
this specific DNA of certain sequences to be isolated. This system
has a double advantage as it is able not only to reveal DNA-protein
interaction in vivo in yeast, but also to give direct access to
complementary DNAs (cDNA) coding for the candidate proteins having
a transcription factor activity. This system is mainly based on the
construct of a test yeast strain according to the principle
developed by Wang and Reed (1993). This yeast strain enables DNAc
banks to be screened by demonstrating DNA-protein interaction in
vivo through activation of a reporter gene integrated within the
genome of the test yeast.
BRIEF SUMMARY OF THE INVENTION
[0017] The object of the present invention is therefore an isolated
polypeptide designated as ICBP90 (inverted CCAAT box binding
protein) with the amino acid sequence SEQ ID No.2. This sequence
comprises: [0018] a) a "ubiquitin" domain comprising the sequence
of amino acids 1-75 of sequence SEQ ID No.2; [0019] b) a "zinc
finger" domain of the C4HC3 type comprising the sequence of amino
acids 310-366 of sequence SEQ ID No. 2 and a "zinc finger" domain
of the C3HC4 type comprising the sequence of amino acids 724-763 of
sequence ID No.2; [0020] c) a presumed "zipper leucine" domain
comprising the sequence of amino acids 58-80 of sequence SEQ ID
No.2; [0021] d) two potential nuclear localization domains
comprising the sequences of amino acids 581-600 and 648-670 of
sequence SEQ ID No.2; [0022] e) a site for phosphorylation with a
tyrosine kinase comprising the sequence of amino acids 452-458 of
sequence SEQ ID No.2; [0023] f) sites for phosphorylation with a
dependent cAMP/cGMP protein kinase comprising the sequences of
amino acids 246-249, 295-298 and 648-651 of sequence SEQ ID No.2;
[0024] g) sites for phosphorylation with a casein kinase II
comprising the sequence of amino acids 23-36, 57-60, 91-94,
109-112, 165-168, 265-268, 354-357 and 669-672 of sequence SEQ ID
No.2; [0025] h) sites for phosphorylation with a protein kinase C
comprising the sequence of amino acids 82-84, 104-106, 160-162,
173-175, 251-253, 301-303, 380-382, 393-395, 504-506, 529-531,
625-627 and 639-641 of sequence SEQ ID No.2.
[0026] The present invention also relates to an isolated
polypeptide characterized in that, it comprises a polypeptide
selected from: [0027] a) a polypeptide of sequence SEQ ID No.2, SEQ
ID No.4, SEQ No.6 or SEQ ID No.8; [0028] b) a polypeptide, a
polypeptide variant of sequences of amino acids defined under a);
[0029] c) a polypeptide homologous to the polypeptide defined under
a) or b) and including at least 80% homology, preferably 90% with
said polypeptide of a); [0030] d) a fragment of at least 5
consecutive amino acids of a polypeptide defined under a), b) or
c); [0031] e) a biologically active fragment of a polypeptide
defined under a), b) or c).
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0032] FIG. 1 illustrates the expression of protein ICBP90 in HeLa
cells (tumor cells) and in pulmonary fibroblasts in primary culture
(non-tumoral cells).
[0033] FIG. 2 illustrates immunoprecipitation of the endogenous
protein.
[0034] FIG. 3 illustrates nuclear localization of the endogenous
protein.
[0035] FIG. 4 illustrates expression of endogenous ICBP59 in
proliferating cells.
[0036] FIG. 5 illustrates expression of ICBP-59 in diverse human
tissues.
[0037] FIG. 6 illustrates nucleotide sequence of ICBP90 (nucleotide
sequence SEQ ID NO. 1).
[0038] FIG. 7 illustrates protein sequence of ICBP90 (amino acid
sequence SEQ ID NO. 2).
[0039] FIG. 8 illustrates detection of ICBP90 in the sera of
patients displaying elevated serum markers for solid tumors.
[0040] FIGS. 9 a-b illustrate structural organization of the ICBP
90 gene. FIG. 9 A. Exons are represented by the boxes: the grey
boxes represent coding exons; white boxes represent non-coding
exons. FIG. 9B is sequence of the 5' flanking region of the ICBP
gene (SEQ ID NO. 12).
[0041] FIG. 10 illustrates analysis of the ICBP promoter.
[0042] FIG. 11 illustrates Northern and Western blot analysis of
the expression of ICBP90.
DETAILED DESCRIPTION OF INVENTION
[0043] It should be understood that the invention relates to
polypeptides obtained through purification from natural sources or
else obtained through genetic recombination or even by chemical
synthesis and they may then include non natural amino acids.
[0044] In the present specification, the term "polypeptide" will be
used for also designating a protein or a peptide.
[0045] The term "polypeptide variant" shall be understood as
designating all the mutated polypeptides which may exist in nature,
in particular in the human being, and which notably correspond to
truncations, substitutions, deletions and/or additions of amino
acid residues. The homologous polypeptides according to the
invention at least retain a domain selected from the DNA binding
domain and/or the interaction domain with another protein.
[0046] It shall be understood that the term "homologous
polypeptide" designates polypeptides having certain modifications,
as compared with the natural polypeptide ECBP90, as in particular a
deletion, addition or substitution of at least one amino acid, a
truncation, an extension and/or a chimeric fusion. Among the
homologous polypeptides, those for which the sequence of amino
acids have at least 80% homology, preferably 90%, more preferably
95%, and most preferably 97% homology with the sequences of amino
acids of the polypeptides according to the invention, are
preferred. In the case of a substitution, one or several
consecutive or non consecutive amino acids are replaced with
"equivalent" amino acids. Here, the expression "equivalent" amino
acid aims at designating any amino acid capable of being
substituted for one of the amino acids of the basic structure
without however changing the essential functional properties or
characteristics, such as their biological activities, of the
corresponding polypeptides such that induction in vivo of
antibodies capable of recognizing the polypeptide for which the
amino acid sequence is comprised within the amino acid sequence SEQ
ID No.2, or in one of its fragments as defined above, and notably
the sequence of amino acids SEQ ID No.4, SEQ ID No.6 and SEQ ID
No.8. These equivalent amino acids may be determined either by
relying on their structural homology with the amino acids which
they replace, or on the results of cross biological activity tests
which may take place for the different polypeptides. As an example,
the possibilities of substitutions which may carried out without
their resulting a deep change in the biological activities of the
corresponding modified polypeptides will be mentioned, for example
replacements of leucine with valine or isoleucine, of aspartic acid
with glutamic acid, of glutamine with asparagine, of arginine with
lysine etc., the reverse substitutions may naturally be
contemplated under the same conditions.
[0047] It shall be understood that the term "biologically active
fragment" designates in particular a fragment of an amino acid
sequence of a polypeptide according to the invention having at
least one of the functional characteristics or properties of the
polypeptides according to the invention, notably in that: (i) it is
capable of being recognized by a specific antibody of a polypeptide
according to the invention; (ii) it has at least one of the domains
or regions as defined above; (iii) it is capable of binding to DNA
and notably to the CCAATT and/or inverted CCAAT boxes; (iv) it is
capable of modulating the expression rate of the gene of
topoisomerase II.alpha., (v) it is capable of modulating cell
proliferation.
[0048] It is understood that the term "polypeptide fragment"
designates a polypeptide including a minimum of 5 amino acids,
preferably 7 amino acids, more preferably 10, and most preferably
15 amino acids. Fragments of a polypeptide according to the
invention, obtained by cleaving said polypeptide with a proteolytic
enzyme, with a chemical reagent, or even by placing said
polypeptide in a very acid environment, are also part of the
invention.
[0049] The polypeptide according to the invention may also be
associated with other polypeptides through protein-protein
interactions. It is understood that the term "protein-protein
interactions" designate associations which directly bring into
contact at least two proteins. Thus, the polypeptide of the
invention may dimerize in order to form homodimers or heterodimers,
or be associated as homomultimers or heteromultimers. The
polypeptide according to the invention may also interact with
another polypeptide in order to exert its action; hence, the
polypeptide according to the invention may also have, in addition
to its DNA binding domain, a domain acting on the transcription
which exerts its action via protein-protein interactions with other
protein components of the transcriptional machinery. It is
understood that the term "protein component of the transcriptional
machinery" designates all transcription factors required for
performing and controlling the transcription reaction.
[0050] The polypeptide according to the invention is characterized
in that it is capable of binding to a DNA sequence and in that it
includes at least a DNA binding domain selected from the group
consisting of a "zinc-finger" domain and a "leucine zipper" domain;
the DNA sequence to which binds said polypeptide is a CCAAT box,
preferably an inverted CCAAT box: ICB.
[0051] It is understood that the term "binding to a DNA sequence",
designates a specific interaction between the polypeptide of the
invention and a DNA sequence by means of a series of weak bonds
formed between the amino acids of the protein and the bases. The
polypeptide according to the invention, has at least a DNA binding
domain which contains at least one of the known protein units
capable of interacting with DNA, i.e. the zinc-finger structure
with which is associated a zinc atom (zinc-finger) the
helix-turn-helix structure, the helix-loop-helix structure, and the
leucine-zipper structure.
[0052] It is understood that the term "zinc-finger unit" designates
a sequence of about twenty amino acids assuming a zinc-finger shape
in space. There are two types of them: those which contain four
cysteines (C4) and those which contain two cysteines and two
histidines (C2H2). These amino acids define the nature of the
zinc-finger and they are located at its base and a Zn.sup.++ ion is
located in the middle of the square formed by these four amino
acids. The polypeptide according to the invention potentially has
two units of type C4.
[0053] It is understood that the term "leucine zipper type units"
designates units belonging to dimeric transcription factors which
are either homodimers or heterodimers. The monomer consists of a
sequence with a basic character which interacts with DNA in a
specific way and of a a helix hydrophobic domain which interacts
with the homologous domain of the other chain. In this domain,
leucine is found every 7 amino acids, i.e. at each turn of the
helix. All these leucines are aligned and the interaction occurs at
their level between both monomers. The polypeptide according to the
invention potentially has a leucine zipper type unit.
[0054] The invention also relates to an isolated polynucleotide
characterized in that it codes for a polypeptide of sequence SEQ ID
No.1 as defined earlier. Preferably, the polynucleotide according
to the invention has the SEQ ID No.1 sequence.
[0055] The invention also relates to the isolated polynucleotide
characterized in that it comprises a polynucleotide selected from:
[0056] a) a polynucleotide with sequence SEQ ID No.1, SEQ ID No.3,
SEQ ID No.5 or SEQ ID No.7 or for which the sequence is that of the
RNA corresponding to sequence SEQ ID No1, SEQ ID No.3, SEQ No.5 or
SEQ ID No.7; [0057] b) a polynucleotide for which the sequence is
complementary to the sequence of a polynucleotide defined under a),
[0058] c) a polynucleotide for which the sequence includes at least
80% homology with a polynucleotide defined under a) or b), [0059]
d) a polynucleotide which hybridizes under high. stringency
conditions with a polynucleotide sequence defined under a), b) or
c), [0060] e) a fragment of at least 15 consecutive nucleotides,
preferably 21 consecutive nucleotides, and more preferably 30
consecutive nucleotides of a polynucleotide defined under a), b),
c) or d), except for human EST AI084125, except for the sequence
corresponding to sequence SEQ ID No.944 published on Aug. 5, 1999
in Patent Application WO 99 38972 and except for sequences SEQ ID
No.9, No.10 and No.11 corresponding to the human ESTs No. AI
0830773, No. AA 811055, No. AA 488 755, No. AA 129 794 and No. AA
354 253 present in the human EST data bases (human dbest),
respectively.
[0061] In the present specification, it is understood that the
terms, "polynucleotide, oligonucleotide, polynucleotide sequence,
nucleotidic sequence, or nucleic acid", shall designate a DNA
fragment, as well as a double strand DNA, a single strand DNA, as
well as transcription products of said DNAs, and/or an RNA
fragment, said isolated natural or synthetic fragments whether
including non-natural nucleotides or not, designating a specific
chaining of nucleotides, whether modified or not, providing
definition of a fragment or a region of a nucleic acid.
[0062] It is understood that the term "polynucleotide" with a
complementary sequence, designates any DNA for which the
nucleotides are complementary to those of SEQ ID No.1, SEQ ID No.3,
SEQ ID No.5, SEQ ID No.7 or of a part of SEQ ID No.1, SEQ No.3, SEQ
ID No.5, SEQ ID No.7 and for which the orientation is inverted.
[0063] In the sense of the present invention, it is understood that
the term "homology percent" designates a percentage of identity
between bases of two polynucleotides, this percentage being purely
statistical and the differences between both polynucleotides are
randomly distributed throughout their length. According to the
invention, the polynucleotides with a homologous nucleic sequence
have a homology rate of at least 80%, preferably 90%, more
preferably 95%, most preferably 97%.
[0064] Hybridization under strong stringency conditions means that
the temperature and ionic force conditions are selected in such a
way that hybridization between two complementary DNA fragments may
be maintained. As an illustration, strong stringency conditions of
the hybridization step for the purpose of defining the
polynucleotidic fragments described above, advantageously are the
following:
[0065] DNA-DNA or DNA-RNA hybridization is achieved in two steps:
(1) prehybridization at 42.degree. C. for 3 hours in phosphate
buffer (20 mM pH 7.5) containing 5.times. SSC (1.times. SSC
corresponds to a 0.15 M NaCl+0.015 M sodium citrate solution), 50%
formamide, 7% sodium dodecylsulfate (SDS), 10.times. Denhard's, 5%
dextran sulfate and 1% salmon sperm DNA; (2) the actual
hybridization for 20 hours at a temperature depending on the size
of the probe (i.e. 42.degree. C., for a probe with a size >100
nucleotides), followed by two washings for 20 minutes at 20.degree.
C. into 2.times. SSC+2% SDS, one washing for 20 minutes at
20.degree. C. into 0.1.times. SSC+0.1% SDS. The last washing is
performed in 0.1.times. SSC+0.1% SDS for 30 minutes at 60.degree.
C. for a probe with a size >100 nucleotides. The strong
stringency hybridization conditions described above, for a
polynucleotide with a defined size, will be adapted by one skilled
in the art for oligonucleotides with a larger or smaller size,
according to the teaching of Sambrook et al., 1989.
[0066] Advantageously, a nucleotidic fragment meeting the earlier
definition will have at least 15 consecutive nucleotides,
preferably at least 21 nucleotides, and even more preferably at
least 30 consecutive nucleotides of the sequence from which it
stems.
[0067] It is understood that the term EST ("expressed sequence
tag") designates expressed sequences, characterized in a
complementary DNA bank (DNAc) and used as a map marker for genomic
DNA.
[0068] According to one embodiment of the invention, the
polynucleotide according to the invention is characterized in that
it is directly or indirectly labeled with a radioactive compound or
a non-radioactive compound. Use of a polynucleotide according to
the invention as a primer for amplifying or polymerizing nucleic
sequences; the invention also relates to the use of a
polynucleotide according to the invention as a probe for detecting
nucleic sequences. According to the invention, the polynucleotide
fragments may be used as a probe or as a primer in methods for
detecting, identifying, dosing and amplifying nucleic sequences,
and they have a minimum size of 9 bases, preferably 18 bases, and
more preferably 36 bases. Finally, the invention is related to the
use of a polynucleotide according to the invention as a sense or
anti-sense nucleic acid sequence for controlling the expression of
the corresponding protein product.
[0069] The non-labeled sequences of polynucleotides according to
the invention may directly be used as a probe, a primer or an
oligonucleotide; however the used sequences are generally labeled
for obtaining usable sequences for many applications. The labeling
of primers, probes, oligonucleotides according to the invention is
achieved through radioactive elements or through non-radioactive
molecules; .sup.32P, .sup.33P, .sup.35S, .sup.3H, or .sup.125I may
be mentioned among the used radioactive isotopes. The
non-radioactive entities are selected from ligands such as biotin,
avidin, streptavidin, dioxygenin, haptenes, dyes, luminescent
agents, such as radioluminescent, chemiluminescent, bioluminescent,
fluorescent, phosphorescent agents.
[0070] The polynucleotides according to the invention may thus be
used as a primer and/or a probe in methods notably implementing the
PCR (polymerase chain reaction) technique (Erlich, 1989; Innis et
al., 1990, and Rolfs et al., 1991). This technique requires the
selection of pairs of oligonucleotidic primers framing the fragment
which should be amplified. Reference may for example, be made to
the technique described in the U.S. Pat. No. 4,683,202. The
amplified fragments may be identified, for example after agarose
gel or polyacrylamide electrophoresis or after a chromatographic
technique like gel filtration or ion exchange chromatography. The
specificity of the amplification may be controlled by molecular
hybridization by using as a probe, nucleotidic sequences of
polynucleotides of the invention, plasmids containing these
sequences or their amplification products. Amplified nucleotidic
fragments may be used as reagents in hybridization reactions in
order to demonstrate the presence, in a biological sample, of a
target nucleic acid with a sequence complementary to that of said
amplified nucleotidic fragments.
[0071] The invention is also directed to nucleotidic fragments
which may be obtained through amplification by means of primers
according to the invention.
[0072] Other techniques for amplifying the target nucleic acid may
advantageously be used as an alternative to PCR (PCR-like) by means
of a pair of primers for nucleotidic sequences according to the
invention. It is understood that the term "PCR-like" designates all
methods implementing direct or indirect reproductions of nucleic
acid sequences, or else those in which the labeling system has been
amplified, of course these techniques are known, generally this
deals with DNA amplification by a polymerase; when the original
sample is an RNA, a reverse transcription should be performed
beforehand. Presently, there are very many methods which provide
such amplification, such as for example, the SDA (Strand
Displacement Amplification) technique (Walker et al., 1992), the
TAS (Transcription-based Amplification System)technique described
by Kwoh et al., in 1989, the 3SR (Self-Sustained Sequence
Replication) technique described by Guatelli et al., in 1990, the
NASBA (Nucleic Acid Sequence Based Amplification) technique
described by Kievitis et al., in 1991, the TMA (Transcription
Mediated Amplification) technique, the LCR (Ligase Chain Reaction)
technique described by Landegren et al., in 1988, and enhanced by
Barany et al., in 1991, which uses a thermostable ligase, the RCR
(Repair Chain Reaction) technique described by Segev in 1992, the
CPR (Cycling Probe Reaction) technique described by Duck et al., in
1990, the Q-beta-replicase amplification technique described by
Miele et al., in 1983, and notably enhanced by Chu et al., in 1986
and Lizardi et al., in 1988, and then by Burg et al., as well as
Stone et al., in 1996.
[0073] If the target polynucleotide is an RNA, for example a RNAm,
a reverse transcriptase type enzyme will advantageously be used
before implementing an amplification reaction with the primers
according to the invention or before implementing a detection
method with probes of the invention, in order to obtain a DNAc from
the RNA contained in the biological sample. The obtained DNAc will
then be used as a target for the primers or the probes implemented
in the detection or amplification method according to the
invention.
[0074] The nucleotidic probes according to the invention,
specifically hybridize with a DNA or RNA polynucleotide molecule
according to the invention, more particularly with the sequence SEQ
ID No.1 coding for the ECBP90 polypeptide, under strong stringency
hybridization conditions such as those given as an example
earlier.
[0075] The hybridization technique may be used in different ways
(Matthews et al., 1988). The most general method consists of
immobilizing the nucleic acid extracted from cells of different
tissues or from cells cultivated on a support (such as
nitrocellulose, nylon, polystyrene) and of incubating, under well
defined conditions, the immobilized target nucleic acid with the
probe. After hybridization, the probe excess is removed and the
formed hybrid molecules are detected by the suitable method
(measurement of radioactivity, fluorescence or enzyme activity
related to the probe).
[0076] According to another embodiment of the nucleic probes,
according to the invention, the latter may be used as a capture
probe. In this case, a so-called "capture probe" is immobilized on
a support and is used for capturing through specific hybridization,
the target nucleic acid obtained from the biological sample to be
tested and the target nucleic acid is then detected by a second
probe, a so-called "detection probe", labeled with an easily
detectable element.
[0077] In a preferred embodiment, the invention comprises the use
of a sense or anti-sense oligonucleotide for controlling the
expression of the corresponding protein product. Among the
interesting nucleic acid fragments, anti-sense oligonucleotides
i.e. those for which the structure provides an inhibition of the
expression of the corresponding product, by hybridization with the
target sequence, may be mentioned in particular. The sense
oligonucleotides which, through interaction with the proteins
involved in the control of the expression of the corresponding
product which will induce either an inhibition, or an activation of
this expression, should also be mentioned. The oligonucleotides
according to the invention, have a minimum size of 9 bases,
preferably 18 bases, and more preferably 36 bases.
[0078] The invention relates to a recombinant vector for cloning a
polynucleotide according to the invention and/or for expressing a
polypeptide according to the invention characterized in that, it
contains a polynucleotide according to the invention, as described
earlier. The vector according to the invention, is characterized in
that it includes components for the expression, possibly the
secretion, of said sequences in a host cell. These vectors are
useful for transforming host cells in order to clone or express
nucleotidic sequences of the invention. Particular vectors are for
examples the vectors of plasmidic or viral origin. Among these
vectors, those of the pGEX series (Pharmacia) for expression in
bacteria or pSG5 (Stratagene, La Jolla, Calif. USA) are preferred
for expression in a eukaryotic system.
[0079] According to a particular embodiment, the vector according
to the invention includes components for controlling expression of
the polypeptides, these control components are preferably selected
from (i) the promoter sequence of the ICBP90 gene according to the
invention which corresponds to sequence SEQ ID No.12; (ii) a
polynucleotide for which the sequence is complementary to the
sequence SEQ ID No.12; (iii) a polynucleotide for which the
sequence includes at least 80% identity with a polynucleotide as
defined in (i) or (ii); (iv) a polynucleotide which hybridizes
under strong stringency conditions with the polynucleotide sequence
defined under (i), (ii), (iii). Computer tools available to one
skilled in the art will easily allow him/her to identify the
required and sufficient promoter control boxes for controlling the
genic expression, notably the TATA, CCAAT, GC boxes, as well as
enhancer or silencer control sequences which control in CIS the
expression of genes according to the invention.
[0080] The use of the above components defined and selected from
the sequence SEQ ID No.12 for controlling the expression of
heterologous polypeptides other than those of the invention and
notably for controlling the expression of heterologous polypeptides
in cell types in which the polypeptides according to the invention
are expressed normally, is also within the scope of the
invention.
[0081] The invention further comprises host cells, notably
eukaryotic and prokaryotic cells, characterized in that they are
transformed with vectors according to the invention. Preferably,
the host cells are transformed under conditions allowing a
recombinant polypeptide according to the invention to be expressed.
The cell host may be selected from bacterial cells (Olins and Lee,
1993), but also from yeast cells (Buckholz, 1993), as well as
animal cells, in particular mammal cell cultures (Edwards and
Aruffo, 1993), but also insect cells wherein methods implementing
baculoviruses for example may be used (Luckow, 1993). These cells
may be obtained by introducing into the host cells a nucleotidic
sequence inserted in a vector such as defined above, and then by
growing said cells under conditions providing replication and/or
expression of the transfected nucleotidic sequence.
[0082] The invention also relates to a method for preparing a
polypeptide, characterized in that it implements a vector according
to the invention. More specifically, the invention relates to a
method for preparing a recombinant polypeptide characterized in
that the transformed cells according to the invention are grown
under conditions providing expression of said recombinant
polypeptide and in that said recombinant polypeptide is
recovered.
[0083] The polypeptide according to the invention may be obtained
according to a method of the invention, and according to production
techniques for recombinant polypeptides, known to one skilled in
the art. The present invention therefore relates to the recombinant
polypeptide which may be obtained by the method shown above. In
this case, the nucleic acid sequence used is placed under the
control of signals providing its expression in a cell host. An
efficient production system for a recombinant polypeptide requires
the availability of a vector, for example of plasmidic or viral
origin and of a compatible host cell. The vector should include a
promoter, signals for initiating and terminating the translation,
as well as suitable regions for controlling the transcription. It
should be able to be maintained in the cell stably and may
optionally have particular signals specifying the secretion of the
translated polypeptide. These different control signals are
selected depending on the used host cell. For this purpose, the
nucleic acid sequences according to the invention may be inserted
in autonomous replication vectors inside the selected host or
integrative vectors of the selected host. Such vectors are prepared
according to methods currently used by one skilled in the art and
the resulting clones may be introduced into a suitable host by
standard methods such as for example transfection with calcium
phosphate precipitation, lipofection, electroporation, thermal
shock.
[0084] The recombinant polypeptides obtained as indicated above,
may both exist in the glycosylated and non-glycosylated form and
may have the natural tertiary structure or not.
[0085] The polypeptides obtained through chemical synthesis and
which may include non-natural amino acids corresponding to said
recombinant polypeptides, are also comprised in the invention. The
peptides according to the invention may also be prepared by
conventional techniques, in the field of peptide synthesis. This
synthesis may be carried out in a homogenous solution or in the
solid phase.
[0086] The methods used for purifying recombinant polypeptides are
well known to one skilled in the art. The recombinant polypeptide
may be purified from lysats and cell extracts, from the supernatant
of the culture medium, by methods either used individually or in
combination, such as fractionation, chromatography methods,
immuno-affinity techniques by means of specific mono- or polyclonal
antibodies, etc.
[0087] A preferred alternative consists of producing a recombinant
polypeptide fusioned to a "carrier" protein (chimeric protein). The
advantage of this system is that it provides stabilization and a
reduction in the proteolysis of the recombinant product, an
increase in the solubility during renaturation in vitro and/or a
simplification of the purification when the fusion partner has an
affinity for a specific ligand.
[0088] The invention also relates to a monoclonal or polyclonal
antibody and to its fragments, characterized in that they
specifically bind a polypeptide according to the invention.
Chimeric antibodies, humanized antibodies and simple
chain-antibodies are also part of the invention. Antibody fragments
according to the invention are preferably Fab or F(ab')2
fragments.
[0089] The polypeptides according to the invention allow monoclonal
or polyclonal antibodies to be prepared. Advantageously, monoclonal
antibodies may be prepared from hybridomas according to the
technique described by Kohler and Milstein in 1975. The inventors
use this technique for obtaining a hybridoma producing a new highly
specific monoclonal antibody of an epitope of protein ICBP90.
[0090] Polyclonal antibodies may be prepared, for example, by
immunizing an animal, for example a mouse, with a polypeptide
according to the invention associated with an adjuvant from the
immune response, and then by purifying the specific antibodies
contained in the serum of the immunized animals on an affinity
column on which is fixed beforehand the polypeptide which has been
used as an antigen. The polyclonal antibodies according to the
invention may also be prepared by purification on an affinity
column, on which a polypeptide according to the invention has been
immobilized beforehand.
[0091] The invention also relates to a specific monoclonal antibody
of the human ICBP90 protein and capable of inhibiting interaction
between ICBP90 and the DNA sequence onto which protein ICBP90
specifically binds. According to another embodiment, the monoclonal
antibody according to the invention and specific to the human
ICBP90 protein is capable of inhibiting the interaction between
ICBP90 and the proteins with which interacts ICBP90, said proteins
preferably being ICBP90 itself, or proteins from the
transcriptional complex. It is understood that the term "proteins
from the transcriptional complex" designates all proteins
participating in the transcription reaction whether this happens in
the initiation, elongation, or termination of the
transcription.
[0092] The antibodies of the invention may also be labeled in the
same way as described earlier for the nucleic probes of the
invention, and preferably with an enzymatic, fluorescent or
radioactive type labeling.
[0093] Moreover, in addition to their use for purifying
polypeptides, the antibodies of the invention, in particular the
monoclonal antibodies, may also be used for detecting these
polypeptides in a biological sample.
[0094] They thus form a means for analyzing the expression of the
polypeptide according to the invention, for example through
immunofluorescence, labeling with gold, enzymatic
immunoconjugates.
[0095] More generally, the antibodies of the invention may
advantageously be implemented in any situation where the expression
of a polypeptide according to the invention needs to be observed,
and more particularly in immunocytochemistry, in
immunohistochemistry, or in Western blotting experiments.
[0096] Thus, the invention relates to a method for detecting and/or
dosing a polypeptide according to the invention, in a biological
sample, characterized in that it comprises the following steps for
bringing the biological sample into contact with antibodies
according to the invention and then for detecting the formed
antigen-antibody complex. This method may be used in
immunocytochemistry for cell localization of the polypeptide
according to the invention and in immunohistochemistry for
assessing cell proliferation.
[0097] A kit for detecting and/or dosing a polypeptide according to
the invention in a biological sample, is also within the scope of
the invention, characterized in that it comprises the following
components: (i) a monoclonal or polyclonal antibody such as
described earlier; (ii) if necessary, the reagents for forming the
favorable medium for the immunological reaction; (iii) the reagents
for detecting the antigen-antibody complexes produced by the
immunological reaction. This kit is notably useful for conducting
Western blotting experiments; with the latter, control of the
expression of the polypeptide according to the invention may be
investigated starting with tissues or cells. This kit is also
useful for immunoprecipitation experiments in order to notably
detect proteins which interact with the polypeptide according to
the invention.
[0098] Any conventional procedure may be implemented for carrying
out such a detection and/or dosage. As an example, a preferred
method involves immunoenzymatic processes according to the
immunofluorescence or radioimmunological (RIA) ELISA technique or
equivalent.
[0099] The invention also comprises a method for detecting and/or
dosing a nucleic acid according to the invention, in a biological
sample, characterized in that it includes the following steps: (i)
isolation of the DNA from the biological sample to be analyzed, or
obtaining a DNAc from the RNA of a biological sample; (ii) specific
amplification of the DNA coding for the polypeptide according to
the invention by means of primers; (iii) analysis of the
amplification products.
[0100] The invention further comprises a kit for detecting and/or
dosing a nucleic acid according to the invention, in a biological
sample, characterized in that it comprises the following
components: (i) a pair of nucleic primers according to the
invention, (ii) the required reagents for carrying out a DNA
amplification reaction and optionally (iii) a component for
checking the sequence of the amplified fragment, more particularly
a probe according to the invention.
[0101] The invention also comprises a method for detecting and/or
dosing a nucleic acid according to the invention, in a biological
sample, characterized in that it includes the following steps: (i)
bringing a probe according to the invention into contact with a
biological sample; (ii) detecting and/or dosing the hybrid formed
between said probe and the DNA of the biological sample.
[0102] The invention also comprises a kit for detecting and/or
dosing a nucleic acid according to the invention, in a biological
sample, characterized in that it comprises the following
components: (i) a probe according to the invention, (ii) the
reagents required for implementing a hybridization reaction and if
necessary, (iii) a pair of primers according to the invention, as
well as the reagents required for an DNA amplification
reaction.
[0103] The invention particularly relates to methods according to
the invention and described above, for detecting and diagnosing
cell proliferation, and more particularly cell proliferation of
cancerous origin.
[0104] The invention also relates to a method for screening ligands
able to affect the transcriptional activity of a gene, the promoter
of which includes CCAAT and/or inverted CCAAT boxes capable of
binding a polypeptide according to the invention, said method being
characterized in that it includes the following steps for bringing
into contact said polypeptide and one or several potential ligands
in the presence of reagents required for implementing a
transcription or detection reaction and/or a reaction for measuring
transcriptional activity. One of the objects of the invention is
also to provide a kit or package for screening ligands able to
affect the transcriptional activity of a gene, the promoter of
which includes CCAAT and/or inverted CCAAT boxes capable of binding
a polypeptide according to the invention characterized in that it
comprises the following components: (i) a polypeptide according to
the invention; (ii) a ligand; (iii) the reagents required for
implementing a transcription reaction.
[0105] The ICBP90 polypeptide according to the invention has a
nuclear receptor function. It is understood that the term "nuclear
receptor" designates a polypeptide which has the essential
properties of hormone nuclear receptors. This gene superfamily
contains i.a. the retinoic acid nuclear receptors (RAR, RXR, . . .
), steroid hormone nuclear receptors (glucocorticoids,
mineralocorticoids, progesterone, androgen, estrogen), and thyroid
hormone nuclear receptors (T3 hormone). Accordingly, one of the
objects of the present invention is also to provide a method for
screening ligands able to affect the "nuclear receptor" function of
the polypeptide according to the invention. Such a method includes
the steps of: [0106] a) bringing into contact the polypeptide of
the invention and one or several potential ligands in the presence
of required reagents; [0107] b) detecting and/or measuring the
transcriptional activity of a gene, the promoter of which includes
nucleotidic sequences onto which the polypeptide of the invention
may be bound. Preferably, said nucleotidic sequences are CCAAT
and/or inverted CCAAT boxes (ICB).
[0108] Techniques for detecting and/or measuring the
transcriptional activity are known to one skilled in the art. The
Northern blotting and RT-PCR technologies should notably be
mentioned, which may be implemented with polynucleotides of the
invention used as a probe or as a primer, respectively.
[0109] It is understood that the term "ligand" defines all
compounds able to interact with the polypeptide according to the
invention, in order to form a complex able to affect the
transcriptional activity, i.e. to increase, reduce, modulate or
cancel the transcription of a gene under the control of a promoter
containing a DNA sequence to which binds the polypeptide of the
invention.
[0110] Such a ligand is therefore able to have an agonist or
antagonist activity. Among the ligands according to the invention,
the biological molecules which interact with the polypeptide
according to the invention as well as all the synthetic chemical
compounds, should be mentioned. Among these ligands, the antibody
according to the invention as well as an oligonucleotide having an
identity of sequence with the CCAAT and/or inversed CCAAT
nucleotidic sequence should also be mentioned; such a ligand is
able to form an inhibitor of the polypeptide according to the
invention.
[0111] The invention also relates to the ligand which may be
obtained by the previous screening methods.
[0112] It is also understood that the term "ligand" defines any
compound able to bind to the binding DNA sequence for the
polypeptide according to the invention. Such a ligand forms a
competitive inhibitor of the polypeptide according to the invention
for its binding to the DNA sequence.
[0113] Preferably, the biological sample according to the invention
in which detection and dosage is performed, consists of a body
fluid, for example human or animal serum, blood, saliva, lung
mucus, or biopsies. The biological liquid resulting from a
broncho-alveolar washing also obtained during analyses for
diagnosing cancers of the deep airways is also included in the
definition of a biological sample of the invention.
[0114] According to another aspect, the invention relates to a
compound characterized in that it is selected from an antibody, a
polypeptide, a ligand, a polynucleotide, an oligonucleotide, or a
vector according to the invention as a drug, and notably as active
ingredients of a drug: these compounds preferably will be in
soluble form, associated with a pharmaceutically acceptable
carrier. It is understood that the term "pharmaceutically
acceptable carrier" designates any type of carrier usually used in
preparing injectable compositions, i.e. a diluent, a suspension
agent, such as an isotonic or buffered saline solution. Preferably,
these compounds will be administered systemically, in particular
intravenously, intramuscularly, intradermally, or orally. Their
modes of administration, dosages and optimal dosage forms may be
determined according to the criteria generally considered in
establishing a suitable treatment for a patient as for example, the
age or body weight of the patient, the seriousness of his/her
general condition, tolerance to the treatment and ascertained
secondary effects, etc.
[0115] According to another aspect, the invention relates to a
compound, characterized in that it is selected from a polypeptide,
a polynucleotide, an anti-sense polynucleotide, an antibody, a
vector, a cell, a ligand according to the invention as a drug and
notably as active ingredients of a drug; these compounds preferably
will be in soluble form, associated with a pharmaceutically
acceptable carrier. It is understood that the term
"pharmaceutically acceptable carrier" designates any type of
carrier usually used in preparing injectable compositions, i.e. a
diluent, a suspension agent, such as an isotonic or buffered saline
solution. Preferably, these compounds are administered
systemically, in particular intravenously, intramuscularly,
intradermally or orally. Their modes of administration, dosages and
optimal dosage forms may be determined according to criteria
generally considered in establishing a suitable treatment for a
patient such as for example the age or body weight of the patient,
the seriousness of his/her general condition, tolerance to the
treatment and the ascertained secondary effects, etc. When the
agent is a polypeptide, an antagonist, a ligand, a polynucleotide,
for example an anti-sense composition, a vector, it may be
introduced into tissues or host cells by a number of ways,
including viral infection, micro-injection, or fusion of vesicles.
Jet injection for an intramuscular administration as described by
Furth et al. (1992) may also be used. The polynucleotide may also
be deposited on gold micro-particles, and be delivered
intradermally by means of a particle bombardment apparatus, or a
"gene pistol" as described in the literature (see for example Tang
et al. (1992) where gold microprojectiles are coated with the
polynucleotide of the invention, preferably the anti-sense
polynucleotide of the invention, then are bombarded into the skin
cells.
[0116] The compound comprising this invention is used for the
preparation of a pharmaceutic designed to modulate, raise, or
diminish cellular proliferation.
[0117] The invention also has at its foundation a pharmaceutical
composition that can act in the preventive and curative treatment
of cancer and is characterised by a therapeutically effective
quantity of an active compound and a pharmaceutically acceptable
excipient. Using the preferred method of synthesis, this
pharmaceutical composition contains antibodies that serve as
targeting agents; those antibodies are conjugated to at least one
agent selected from among antiproliferative, antineoplastic, or
cytotoxic agents. These agents are either radioisotopes or
non-isotopic substances. The conjugation of antibodies contained in
the present invention with antiproliferative, antineoplastic, or
cytotoxic agents can be utilized for arresting the development of
cancers and for inducing regression and even elimination of
tumoural masses. Preferably, the antibody or the antibody fragment
conjugated to the agent is administered to the cancer patient and
delivered to tumour sites by oral or parenteral route through a
pharmaceutically acceptable transporting liquid, such as saline.
Alternatively, a solution or suspension of antibody and antibody
fragment conjugated to an agent can be perfused directly into the
tissue of a malignant epithelial cancer, a method used by
preference when the cancer has not metastasized.
[0118] For therapeutic use, the preferred radioisotopes, conjugated
to monoclonal antibodies, are gamma emitters, the most effective
being iodine.sup.131, yttrium.sup.90, gold.sup.199,
palladium.sup.100, copper.sup.67, bismuth.sup.217, and
antimony.sup.211. Alpha and beta emitting radioisotopes can also be
employed for therapy. Non-isotopic substances conjugated to
monoclonal antibodies and used for therapy are abundant and varied;
for example: (i) antimetabolites, such as anti-folate agents like
methotrexate, (ii) purine and pyrimidine analogues (mercaptopurine,
fluorouracil, 5-azacytidine, (iii) antibiotics, (iv) lectins
(ricin, abrin) and (iv) bacterial toxins (diphtheria toxin).
[0119] The antibodies of the invention can also be used as
targeting agents to target cytotoxic cells, such as human T cells,
monocytes or NK cells present or not at a metastasised tumour site.
Antibodies can attach to cytotoxic cells via the Fc receptor
situated at the surface of these cells or via an intermediary
antibody that has a double specificity. Such bi-specific antibodies
for the targeting of cancerous cells can be produced by fusing an
immune cell producing the antibody of the present invention or a
hybridoma of the present invention with a cell producing an
antibody directed against the targeted cytotoxic cell. Bi-specific
antibodies can equally be produced by chemically coupling two
antibodies having the desired specificity. The antibodies of this
invention also permit the targeting of carriers bearing
antiproliferative, antineoplastic, or cytotoxic agents to the site
of the tumor or metastatic tumor. By carriers we are referring to
liposomes and viral particles. In certain cases, it's possible to
predetermine the target elements to assure a specific expression in
certain tissues or cells and limit the expression zones of the
polypeptides of this invention.
[0120] The invention also concern a product comprising at least a
compound of the invention, and at least an anticancerous agent as a
combination product for a simultaneous, separated or delayed use
over the time.
[0121] In summary, the invention concerns a composition for the
detection, localisation, and imaging of cancers, using an antibody
that is tagged directly or indirectly by a marker whose signal is
generated by radioactive or non-isotopic substances as defined
above. The invention also has as objective the localisation and
imaging of cancers, including (i) the stages of dispersion after
parenteral injection into a human of a composition based on the
invention; (ii) the accumulation of tagged antibody, after an
adequate time period, at the vicinity of cancer cells, then the
penetration of those cells by the tagged antibody without
significantly affecting normal cells; (iii) the detection of a
signal using an appropriate signal detector; and (iv) the
conversion of the detected signal to an image of the cancerous
cells.
[0122] Other characteristics and advantages of the invention are
discussed after this description accompanied by the examples below.
In the examples, we will refer to the following figures.
FIG. 1: Expression de la protein ICBP90 in HeLa Cells (Tumour
Cells) and in Pulmonary Fibroblasts in Primary Culture (Non-tumoral
Cells).
[0123] The detection of the endogenous protein, ICBP90, was carried
out on total protein extracts from confluent (lane 1) and
proliferating (lane 2) HeLa cells and on total protein extracts
from primary cultures of human pulmonary fibroblasts at confluence
(lane 3) and in proliferation (lane 4). After migration in a
polyacrylamide gel in the presence of 8% SDS, the proteins were
transferred to nitrocellulose membranes by electrotransfer. The
revelation of the protein was performed using antibody lRClC-10
diluted to 1/4000 (initial concentration 2 mg/ml) and a secondary
antibody coupled to alkaline phosphatase and directed against the
heavy chains of mouse antibodies. In the lanes corresponding to
extracts from HeLa cells, there is a major band at 97 kDa; for
proliferating HeLa cells, supplementary bands of sizes less than 97
kDa appear (lane 2). In confluent human pulmonary fibroblasts, the
endogenous protein is not expressed (lane 3), while the protein
does appear when the cells begin to proliferate (lane 4). These
observations suggest that the endogenous ICBP90 protein is a marker
of cellular proliferation for normal cells (fibroblasts), whereas
for tumour cells, it is a marker regardless of the cellular
stage.
FIG. 2: Immunoprecipitation of the Endogenous Protein
[0124] Immunoprecipitation was carried out on total protein
extracts from MOLT-4 cells. 1RC1C-10 antibodies were attached to
the protein beads of G-Sepharose, then put into contact with
protein extract for 2 hours at room temperature. After washing, the
bead/1RC1C-10/protein complexes were precipitated by centrifugation
and analysed by migration in a 8% polyacrylamide gel in the
presence of SDS. They were then transferred to nitrocellulose
membranes for revelation of the proteins as indicated in FIG. 1. A
unique band appears at 97 kDa, as. well as a band of 45 kDa
corresponding to the heavy chain of 1RC1C-10.
FIG. 3: Nuclear Localisation of the Endogenous Protein
[0125] We used HeLa cells to examine the endogenous expression of
the protein ICBP90 in situ employing 1RC1C-10 antibody and a
secondary anti-mouse antibody coupled to fluorochrome CY3. The
fluorescent marker localises exclusively in the nucleus. The
nucleolus and the cytoplasm are not labelled.
FIG. 4: Expression of Endogenous ICBP59 in Proliferating Cells
[0126] We observed endogenous protein in paraffin sections of human
appendix. After deparaffinization and pre-treatment by heat in acid
buffer (unmasking of antigenic sites), the sections were incubated
for 16 hours with 1RC1C-10 antibodies diluted 1/10000 (initial
concentration of 2 mg/ml). Revelation was performed by adding
biotinylated secondary antibody, and then incubating with
streptavidine-peroxidase complex. A counter-staining of nuclei by
Harris' haematoxylin was also carried out. The labelling by
1RC1C-10 is localised essentially in zones of cellular
proliferation. The labelled cells are found in glandular crypts
(GC), as well as germinative zones (ger).
FIG. 5: Expression of ICBP-59 in Diverse Human Tissues
[0127] We evaluated the level of expression of mRNA corresponding
to ICBP59 in 50 different human tissues using an RNA dot blot. The
blot was hybridised for 16 hours at 68.degree. C. with a cDNA (32P)
radioactive probe of 679 bp in ExpressHyb (Clontech) hybridisation
solution. After washing several times, we revealed the protein by
autoradiography (one week exposure at 80.degree. C.). The tissues
demonstrating the highest expression level were foetal and adult
thymus, as well as adult bone marrow and foetal liver.
FIG. 6: Nucleotide Sequence of ICBP90 (Nucleotide Sequence SEQ ID
NO. 1).
[0128] cDNA coding for ICBP90 (nucleotide SEQ ID NO. 1) measures
2379 bp. The portions of sequence indicated in bold are those that
do not appear in the human EST database (human dbest). The other
sequences exist in diverse EST: [0129] From 1 to 325: EST n.degree.
AA083773, [0130] From 367 to 865 EST n.degree. AA811055. [0131]
From 940 to 1857 EST n.degree. AA488755, EST n.degree. AA129794 and
EST n.degree. AA354253. FIG. 7: Protein Sequence of ICBP90 (Amino
Acid Sequence SEQ ID NO. 2).
[0132] The amino acid sequence of ICBP90 (amino acid sequence SEQ
ID NO. 2) was deduced by translation of the nucleotide sequence
from FIG. 6 (SEQ ID NO. 1). ICBP90 (amino acid sequence SEQ ID NO.
2) is composed of 793 residues and has a theoretical molecular
weight of 89,758 kDa. The pKi is 7.7. The amino acids indicated in
grey correspond to ICBP-59.
FIG. 8: Detection of ICBP90 in the Sera of Patients Displaying
Elevated Serum Markers for Solid Tumours.
[0133] A volume of 2 .mu.l of serum from each patient was diluted
in 1 ml of PBS (1X Phosphate Buffered Saline) containing 0.1%
Tween-20 followed by serial dilutions carried out in the same
buffer as indicated in the figure. A 0.5 ml aliquot of each
dilution was filtered onto a nitrocellulose membrane using a "Slot
Blot BioRad" apparatus. The membrane was then blocked in the
presence of PBS buffer (containing 0.1% Tween-20 and 5% milk) for 1
hour at room temperature. The protein ICBP90 was revealed by
1RC-1C10 antibodies (1 ng/ml) and anti-mouse secondary antibodies
coupled to peroxidase diluted by 1/5000. The bands were uncovered
by chemiluminescence (10 second exposure of X-MAT (Kodak)
film).
FIG. 9: Structural Organisation of the ICBP90 Gene.
[0134] A. Exons are represented by the boxes: the grey boxes
represent coding exons; white boxes represent non-coding exons. The
size of each exon is indicated in bp in each box, and the names of
the exons are above the boxes. Introns are illustrated
schematically by fine lines and their approximate sizes are in bp.
A putative transcription start site and a polyadenylation consensus
signal are indicated. The ATG is the start codon marking the
beginning of translation and TGA, the stop codon for the end of
translation.
[0135] B. Sequence of the 5' flanking region of the ICBP90 gene
(Seq ID N.degree. 12) (Genbank Accession N.degree. AF 220 226
submitted Dec. 30, 1999). The exons are uppercase and the introns
are lowercase. The start codon ATG is in bold uppercase, the boxes
rich in GC (GC) and the CCAAT (CB) boxes are in bold lowercase.
FIG. 10: Analysis of the ICBP90 Promoter.
[0136] The promoter sequence of ICBP90 was ligated to the reporter
gene, CAT, contained on the pBLCAT2 vector and subsequently
transfected into COS-1 cells.
[0137] A schematic representation of the constructions appears on
the left, the numbers referring to the nucleotides upstream of the
start codon. Relative CAT activity of cellular extracts compared to
induction of CAT activity by the minimal TK promoter are expressed
in percentage (based on the results of 3 independent transfection
experiments) and indicated on the right.
FIG. 11: Northern and Western blot analysis of the expression of
ICBP90.
[0138] A. Northern hybridisation was performed on a Northern
blotting membrane, containing samples of RNA from cell lines of
cancers and various organs. A specific probe for ICBP90,
synthesized by PCR, and labelled by digoxigenin, was used to detect
ICBP90 mRNA. mRNA sizes are noted on the right side of line 7.
[0139] Lines 1 through 7 represent RNA from, respectively,
leukaemic HL-60 promyelocytes, hela 53 cells K562 cells from
chronic myelogenic leukaemia, MOLT-4 lymphoblastic leukaemia cells,
Raji cells from Burkitt's lymphoma, SW480 cells from colorectal
adenocarcinoma, and A549 cells from pulmonary carcinoma.
[0140] The histogram demonstrates the rate of expression of mRNA
corresponding to 5.1 kb and 4.3 kb bands by percentage of the rate
of mRNA expression of the 5.1 kb band of HL-60 (line 1, FIG.
11A).
[0141] B. Western blot analysis of ICBP90 expression in MOLT-4 and
HeLa cells.
[0142] We prepared total cell lysates from proliferating HeLa and
MOLT-4 cell cultures. The expression of ICBP90 was analysed by
Western blotting using 1RC1C-10 antibodies.
EXAMPLE 1
Evidence of a New Binding Protein for the ICB Sequence
[0143] 1.1 Reporter Construction for the Screening of the
Library
[0144] The simple hybrid system is a powerful technique for
detecting, in vivo, in yeast the interaction of proteins with
specific DNA sequences when screening cDNA libraries. This
technique allows you to evaluate directly cDNA corresponding to the
protein to be linked. Several studies using this technique resulted
in the identification of novel proteins. The protocols are well
described by Inouye et al. (1994) and Wang and Reed (1993).
[0145] Briefly, the following oligonucleotides have been
synthesized: TABLE-US-00001 (SEQ ID NO:15)
5'-AATTCGGGGCGGGGCCGGGGCGGGCCCGGGGCGGGGCT-3' (SEQ ID NO:16)
5'-CTAGAGCCCCGCCCCGGCCCCGCCCCGGCCCCGCCCCGG-3'
[0146] These nucleotides were then hybridised. According to the
documentation of the manufacturer (Clontech, Palo Alto, Calif.),
the reporter construct targeted possesses three copies in tandem of
the ICB2 sequence (ICB2X3). As mentioned above, one copy of ICB2 is
underscored and the CCAAT sequences are in bold. To determine the
specificity of protein binding to the ICB box, the following
oligonucleotides, containing three copies in tandem of the GC1 box
(GC1X3), also present in the promoter, have been synthesized and
hybridised: TABLE-US-00002 (SEQ ID NO:13)
5'-AATTCGATTGGTTCTGATTGGTTCTGATTGGTTCTT-3' and (SEQ ID NO:14)
5'-CTAGAAGAACCAATCAGAACCAATCAGAACCAATCG-3'
[0147] The resulting target DNA fragments were cloned into the
polylinker of the pHISi-1 integrative plasmid (Clontech) by
cohesive-end ligation to the plasmid's XbaI-EcoRI site, upstream of
the minimal promoter of the gene, his3. The yeast strain, YM4271
(Clontech), was used for the transformation. Transformed colonies
of yeast containing the plasmid integrated in their genomes were
selected by cultivating the yeast in synthetic dropout medium
lacking histidine. We isolated two colonies: one for ICB2 and the
other for the GC1 box.
[0148] 1.2 Screening the Library
[0149] A cDNA library from the Jurkat cell line, cloned into the
EcoRI site of the polylinker downstream of GAL4-AD of the pGAD10
vector (Clontech), was used for screening according to the
manufacturer's instructions. Positive clones were selected, and
then cultivated in selective medium depleted of histidine and
leucine. The plasmid DNA of the clones was recuperated and
introduced by electroporation into the bacterial Escherichia coli
strain, XL1-blue. The sequencing of the inserts were carried out on
a matrix of plasmid DNA purified from a 1.5 ml culture using a mini
preparation kit (Bio-Rad, Hercules, Calif., USA). A cDNA library of
human thymus cloned in .lamda.gt10 (Clontech) was screened by
plaque hybridisation to recuperate a cDNA coding for the N-terminal
part of the protein.
[0150] 1.3 Discovery of ICBP-59
[0151] The cDNA from four clones selected using the simple hybrid
system was sequenced, then analysed employing a digital database
(Genbank, EMBL, PDB, Swissprot) to determine the nature of the
coded proteins. Two of the clones correspond to ribosomal proteins
(hRS12 and hRS4), one to a serine-threonine kinase (STPLK-1), and
the fourth to a human protein having theoretical molecular weight
of 59 kDa (calculated from the translated sequence) that does not
appear in the database.
[0152] The cDNA coding for hRS4, hRS12, and ICBP-59, and obtained
by EcoRI digestion of positive clones in the pGAD10 vector, were
cloned into the EcoRI site of the expression vector pGEX-4T-1
(Pharmacia). The recombinant DNA was then transformed in an adapted
mouse Escherichia coli strain (BL21). We then used a 500 ml culture
of a selected clone once the culture reached a density of 0.5. The
overexpression of proteins under study was induced by incubation
with IPTG (1 mM) for 2 hours at 37.degree. C. The pGEX-4T-1 vector
makes possible the recovery of large quantities of proteins fused
to glutathione S-transferase (GST). The GST fusion proteins are
then purified using Sepharose beads coupled to glutathione
(Pharmacia) followed by overnight cleavage with thrombin (0.05
U/ml) at 4.degree. C. (Pharmacia).
[0153] To test the ability of the 59 kDa protein to bind
specifically to the ICB1 and/or ICB2 boxes, three tandem copies of
ICB2 (ICB2X3, sequences described above) were labelled at the
terminal end with 32 P phosphore using the T4 polynucleotide kinase
(New England Biolabs) and [.gamma..sup.32P]ATP (160 mCi/mmol, ICN
Irvine, Calif., USA). To examine the specificity of the binding,
oligonucleotides containing only one copy of the CCAAT box were
synthesized: TABLE-US-00003 ICB1: 5'-AGTCAGGGATTGGCTGGTCTG-'; (SEQ
ID NO:17) 5'-CAGACCAGCCAATCCCTGACT-3' (SEQ ID NO:18) ICB2:
5'-AAGCTACGATTGGTTCTTCTG-3'; (SEQ ID NO:19)
5'-CAGAAGAACCAATCGTAGCTT-3'. (SEQ ID NO:20)
[0154] The ICBP-59 protein (1 .mu.g) was incubated with 1 ng of
oligonucleotide and labelled at its terminal end by phosphorous
.sup.32P in 12% glycerol, 12 mM HEPES-NaOH (pH 7.9), 60 mM KCl, 4
mM Tris-HCl (pH 7.9), 100 ng BSA, 0.6 mM DTT, and 100 ng
poly(dI/dC) in 20 .mu.l (Inouye et al., 1994). After a 30-minutes
incubation at room temperature, the reaction mix was loaded in 6%
polyacrylamide gels. In competition experiments, the quantity
indicated of non-labelled oligonucleotides were added to the
reaction mix 10 minutes before the addition of proteins. To examine
the binding properties of ICBP90 with regard to the ICB2 box, we
used the same protocol except that labelled oligonucleotide
contained only one copy of the CCAAT sequence as described below:
TABLE-US-00004 ICB2: 5'-ATAAAGGCAAGCTACGATTGGTTCTTCTGGACG (SEQ ID
NO:21) GAGAC-3' 5'-GTCTCCGTCCAGAAGAACCAATCGTAGCTTGCC (SEQ ID NO:22)
TTTTAT-3'
[0155] Binding specificity was studied using a non-labelled
nucleotide containing a GC box of the human topoisomerase II.alpha.
promoter: TABLE-US-00005 (SEQ ID NO:23)
5'-GAATTCGAGGGTAAAGGGGCGGGGTTGAGGCAGATGCCA-3' (SEQ ID NO:24)
5'-TGGCATCTGCCTCAACCCCGCCCCTTTACCCTCGAATTC-3'.
[0156] These gel retardation experiments in acrylamide gels has
given us evidence that the new 59 kDa human protein can
specifically bind an ICB DNA sequence. We have called this protein
ICBP-59 (amino acid sequence aa 263 to 793 of the sequence SEQ ID
NO. 2) (for inverted CCAAT Box Binding Protein of 59 kDa).
EXAMPLE 2
Characterisation of the ICBP90 Protein
[0157] 2.1. Synthesis of Antibodies
[0158] Mouse monoclonal antibodies were synthesized in our
laboratory by injection of ICBP-59 protein using traditional
methods (Brou et al., 1993); the protein was purified beforehand by
a fusion GST system. Two monoclonal antibodies from 1RC1C-10 and
1RC1H-12 were selected for their ability to detect the ICBP-59
endogenous protein; their specificity was demonstrated in both
Western blotting and immunocytochemistry experiments. Before use,
the antibodies were purified on a DEAE-cellulose column (DE52,
Whatmann) from ascites fluid.
[0159] 2.2 Detection of the Endogenous Protein by Western
Blotting
[0160] To detect endogenous ICBP-59 protein, we first used 1RC1C-10
in a Western blot (0.4 .mu.g/ml 1RC1C-10 monoclonal antibodies) of
nuclear extracts from confluent and proliferating HeLa cells (FIG.
1). COS-1 and HeLa cells were cultivated as previously described
(Brou et al., 1993; Gaub et al., 1998; Rochette-Egly et al., 1997).
MOLT-4 cells were cultured in 100% air in RPMI supplemented with
10% foetal calf serum. Primary cultures of human pulmonary
fibroblasts were prepared and grown in DMEM/F12 as previously
described (Kassel et al., 1998). We purchased nuclear extracts of
Jurkat cells from Sigma, while we prepared the extracts from MOLT-4
and HL60 as already described in the literature (Lavie et al.1999)
. Proliferating HeLa cells and human pulmonary fibroblasts were
obtained by depleting their culture media of serum for 30 hours,
then reintroducing foetal calf serum to a concentration of 10%
(v/v) for 16 hours. Proliferation was arrested when the cells
reached 60 to 70% confluence. Cell cultures stopped at confluence
(100% confluence) were prepared in the same way, omitting the serum
depletion step. For these two types of cells, total cellular
extracts were prepared by first harvesting the cells in PBS
(phosphate buffered saline), then sonicating them. Immunotransfer
experiments on total cell lysates and nuclear extracts involved
loading the material on 8% SDS polyacrylamide gels and performing a
one-dimensional electrophoresis. The proteins were transferred to
nitrocellulose membranes that had been blocked with 10% blocking
reagent (Roche Molecular Biochemical, Mannheim, Germany). They were
then incubated with 1RC1C-10 purified monoclonal antibodies at a
concentration of 0.5 .mu.g/ml. A sheep anti-mouse antibody coupled
to alkaline phosphatase (fragments Fab, Roche Molecular
Biochemicals) was used at a 1/2500 dilution. The signals were
detected using 4-nitro blue tetrazolium
5-bromo-4-chloro-3-indolyl-phosphate chloride as substrate.
[0161] These experiments show that the endogenous protein has a
molecular weight of approximately 97 kDa. Moreover, we observed
that the form of the protein varies according to its tumoural or
non-tumoural nature, as well as the state of confluence or
proliferation of the cells. For example, in the lanes corresponding
to extracts from HeLa cells, there is a major band at 97 kDa; for
proliferating HeLa cells, supplementary bands of sizes inferior to
97 kDa appear (lane 2). In confluent human pulmonary fibroblasts,
the endogenous protein is not expressed and appears when the cells
begin to proliferate (lane 4). These observations suggest that the
endogenous protein ICBP90 is a marker of cellular proliferation in
normal cells (fibroblasts), while, in tumour cells, it would be a
marker at any cellular stage.
[0162] The use of monoclonal antibodies in immunoprecipitation
experiments on nuclear protein extracts, followed by Western
blotting, further puts in evidence the presence of a 97 kDa protein
(FIG. 2).
[0163] The results obtained from Western blotting, for both nuclear
protein extracts and immunoprecipitations, show that the 59 kDa
protein isolated by the simple hybrid system constitutes a fragment
of the corresponding human endogenous protein, in this case, the
C-terminal fragment from residue D263. It was, therefore, necessary
for us to undertake a new screening of the cDNA library.
[0164] 2.3. Multiple Human Tissues RNA Dot Blot Analysis
[0165] In order to choose a library providing us with the best
possible chance to isolate the complete protein, we wanted to
identify a human tissue expressing the corresponding messenger RNA
(mRNA) With a 32P labelled cDNA probe covering part of the ICBP59
sequence, we tested the mRNA expression of interest in 50 different
human tissues against a RNA Dot Blot. Briefly, a 678 base pair
probe corresponding to the ICBP90 amino acids sequence 269 to 500
was synthesized by PCR using Taq polymerase (Sigma, St Louis, Mo.,
USA). The probe labelled by random priming using dCTP - .alpha. 32P
was purified on Sephadex G50 columns (Pharmacie, Uppsala,
Sweden).
[0166] A multiple organ RNA Dot Blot containing poly(A); RNA from
50 different human tissues was hybridised for 20 hours under strong
stringency conditions in an ExpressHyb environment (Clontech) at
68.degree. C. with a 32P labelled probe. High stringency washing
was completed in 0.1.times. SSC, 0.1% SDS at 68.degree. C. (De
Vries et al., 1996).
[0167] The results obtained (FIG. 5) show that tissues expressing
most strongly the ICBP-59 protein mRNA are adult and foetal thymus,
as well as adult bone marrow and foetal liver. Therefore, to
isolate the whole protein, we choose an adult thymus cDNA
library.
[0168] 2.4. Library Screening and ICBP90 Cloning
[0169] The bank screening permitted us to obtain several clones of
about 4000 base pairs (bp) containing a 2379 bp open reading frame
(FIG. 6). This sequence codes for a 793 amino acid protein (FIG.
7), which theoretical molecular weight (calculated from the
translated sequence) is 89.758 kDa. We called this protein ICBP90
(for Inverted CCAAT Box Binding Protein of 90 kDa) by analogy to
the initial 59 kDa protein name.
[0170] The ICBP90 cDNA (2379 bp) was synthesized by PCR using Deep
Vent DNA polymerase (New England Biolabs, Beverly, Mass., USA) and
oligonucleotides used during this PCR reaction were near the EcoRI
site. The product of the reaction was thereafter sub-cloned in a
pGEX-4T-1 vector (Pharmacie) for the GST fusion protein expression
in BL21. The over expression was induced by IPTG (1 mM) for 4 h at
25.degree. C. The ICBP90 protein was then purified.
[0171] 2.5. Immunocytochemistry and Immunohistochemistry.
[0172] The direct observation of the ICBP90 protein on cells and
tissues was also carried out.
[0173] COS-1 cells were transfected as describes previously (Brou
et al., 1993; Gaub et al., 1998) with the pSG5 vector (Stratagene,
La Jolla, Calif.) in which the ICBP90 cDNA (2379 bp) was sub-cloned
in the EcoRI restriction site. The cDNA was synthesized by
polymerisation chain reaction (PCR) using Deep Vent polymerase (New
England Biolabs) and the oligonucleotides flanking the EcoRI
restriction site. Plasmidic construction was verified by
sequencing. The immunolabelling of the transfected lleLas and COS-1
cells was achieved as described previously (Brou et al., 1993) with
1RC1C-10 and 1 RC1H-12 monoclonal antibodies, respectively. An
indirect labelling with ICBP90 immunoperoxidase and II.alpha.
topoisomerase was achieved as described previously (Rio et al.,
1987, Devys et al., 1993). Human appendices were embedded in
paraffin and fixed in 10% buffered formalin (Sigma). Serial
sections (3 .mu.m) were incubated overnight at room temperature
with 1 RC1C-10 antibody and with II.alpha. anti-topoisomerase
antibody (NeoMarkers, Union City, Calif., USA). Antibodies bound in
a specific manner are visualized through a complex using
streptavidine biotin (LAB/LSAB method, Dako LSAB2 System kit; DAKO,
Carpinteria, Calif., USA).
[0174] In immunocytochemistry the 1RC1C-10 antibody labels the HeLa
cells nucleus whereas the nucleolus and the whole cytoplasm are not
labelled (FIG. 3). In immunohistochemistry, paraffin-embedded human
appendix sections show labelling essentially localized in cellular
proliferation zones (FIG. 4). Indeed, the labelled cells were
located in the glandular crypts (CG) as well as in the germinative
zones (Ger). An identical labelling is obtained when using an
II.alpha. anti-topoisomerase antibody, an enzyme essentially
expressed in proliferating cells (results non illustrated).
[0175] 2.6. BLAST Research and Domain Prediction
[0176] Studies about on-line BLAST have been carried out based on
information from the National Centre for Biotechnology Information
at the National Institute of Health (Bethesda, Md., USA).
SCANPROSITE and PROFILESCANS were used for protein analysis
(Infobiogen, Villejuif, France).
[0177] ICBP90 includes a "ubiquitin like" domain in its first 80
amino acids, two sites of potential nuclear localizations in its C
terminal and two zinc finger-like domains, one of which could be
implicated in the DNA linkage and the other in protein-protein
interactions. Several potential phosphorylation sites by protein
kinase C, the casein kinase II, as well as by a tyrosine kinase,
were also present.
[0178] ICBP90 production and purification using the GST fusion
system (same procedure as for ICBP-59) permitted to finally test
the complete protein ability to link the ICB type DNA sequences.
Its behaviour is identical from top to bottom to that observed for
ICBP-59.
[0179] Finally, we isolated a new human protein that we called
ICBP90 for the reasons evoked above. Its theoretical molecular
weight is 89.758 kDa and its apparent molecular weight on
acrylamide gel is 97 kDa. This protein is not only localized
exclusively in human cell nuclei, but it also presents the ability
to bind specifically DNA sequences, in this case CCAAT type
sequences. For these reasons, we think that ICBP90 has the
possibility to modulate the expression of genes which promoter is
provided with CCAAT boxes, possibly in reversed position (ICB). The
gene of the human topoisomerase II.alpha. we are especially
interested in, and which includes five ICB sequences in its
promoter, seems to be one of ICBP90 privileged targets.
[0180] These experiences allowed to bring to light the 1RC1C-10
antibody remarkable features, which only labels proliferating cells
in the case of non cancerous cells; it labels both proliferating
and quiescent cancerous cells; it is usable with 4 different
techniques (Western blotting, Immunocytochemistry, immunohistology,
immunoprecipitation); it has a very good affinity and allow for the
use of 1/150,000 dilution in immunohistochemistry (13 ng/ml);
finally, its use generates nearly no background noise.
[0181] Future applications of 1RC1C-10 are primarily for diagnostic
and basic research. For anatomo-pathologic diagnostics for
instance, it would be quite possible to assess the proliferative
state of a given cancerous tissue. Regarding basic research,
investigations are in progress in our laboratory in order to
determine the exact contribution of ICBP90 to proliferation
mechanisms in normal and cancerous cells. However, the use of
antibodies will be required to study ICBP90 expression as a
function of the cellular cycle, of its precise nuclear localization
and of its interaction with other cellular proteins.
[0182] At the moment we haven't study the expression of ICBP90 with
regards to cellular cycle. Nevertheless, in the case where
cancerous cell lineages are confluent or when they are not
proliferating, we can detect significant differences of ICBP90
expression (FIG. 1) at least with regard to the 97 kDa form. On the
other hand, in the non-cancerous confluent cells (human bronchial
smooth muscular cells) the ICBP90 expression is hard to detect
(results not illustrated). This was confirmed with histological
sections where no quiescent cells were labelled by the antibody. It
is therefore possible that ICBP90 is expressed whatever the
cellular cycle phase in cancerous cells whereas its expression
would vary according to each phase in non-cancerous cells.
Therefore, this makes the use of the antibody extremely
interesting, as, contrary to other cellular proliferation label
such as Ki-67, topoisomerase II.alpha., cycline E and cycline B1,
we would have at our disposition a label for cancerous tissue
proliferating cells that would not depend on the cellular cycle
phase. Indeed, the end of the S phase is characterized by a very
weak Ki-67expression, cycline E labels cells at the end of phase G1
up to the middle of phase S, and cycline B1 labels cells in phase
G2/M (for a review, see Darzynkiewicz et al., 1994). Moreover, it
has been shown that PCNA (Proliferating Cell Nuclear Antigen)
overestimates the number of proliferating cells in some types of
tissues (Roskell and Biddolph, 1999).
[0183] ICBP90 plays an important role in cellular proliferation by
regulating the expression of genes such as those for topoisomerase
II.alpha.. Different strategies aiming at blocking the action of
this protein must allow modifying cellular proliferation. Anyway,
the uses of the 1RC1C-10 antibody as well as of peptides mimicking
the ADN/ICBP90 interaction without generating subsequent
physiological effect constitute an interesting possibility. The
design of its peptides would be directly inspired from the ICBP90
protein sequence we described. A truncated form corresponding to
ICBP59 could be one of the first candidates, for instance.
[0184] The simple blockage of ICBP90 expression in order to
completely eliminate its influence on genes and, by extension, on
cellular proliferation can be considered; it could be carried out
either by a classic approach such as obtaining inhibitors of the
protein, or by a more modern approach corresponding to the
interference technique with-double strand RNA (RNA interference or
RNAi) as describes recently by Kennerdell & Carthew (1998).
EXAMPLE 3
Isolation and Characterization of Gene ICBP90
[0185] 3.1. Material and Methods
[0186] 3.1.1. Construction and Screening of a Human Placental
Genomic Library
[0187] After partial digestion with MboI enzyme, the placental
genomic DNA was split up according to size on a 10 to 40% sucrose
gradient. Fifteen kb DNA Fragments were ligated in a .lamda.GEM 12
vector previously digested with BamHI (Promega, Madison Wis., USA).
After packaging, phage .lamda. particles were assayed on TAP 90
cells. The genomic library contains 3.106 plaque-forming units
(pfu). 10.sup.6 clones were spread out for analysis. A 620 bp probe
corresponding to a 5' terminal extremity of the ICBP90 cDNA used
for the screening was labelled with .alpha..sup.32P-dCTP by a
random priming method (Sambrook et al., 1989). The labelled probe
is used according to a classic on plaque hybridisation protocol to
screen the genomic library (Sambrook et al., 1989). Hybridisation
was achieved at 68.degree. C. in 5.times. SSC (15 mM NaCl, 1.5 mM
sodium citrate pH 7.0), 5 .times. Denhardt solution, 100 .mu.g/ml
of salmon sperm DNA, and 0.1% SDS, followed by 30 minutes washing
in 2.times. SSC, 0.1% SDS at room temperature.
[0188] Two screening steps were completed to purify a positive
clone. The positive clone was then digested with NotI enzyme and
two fragments of 6 and 10 kb were sub-cloned in pBluescript
SK+vector (Stratagene, La Jolla Calif., USA) following a standard
protocol (Sambrook et al., 1989).
[0189] 3.1.2. Library Screening of Human Thymus cDNA
[0190] A bank AGT10 of human thymus cDNA 5' end (Clontech, Palo
Alto, Calif., USA) has been screened by on plaque hybridisation
using the 679 bp cDNA probe synthesized as in the paragraph
concerning Northern Blotting Analysis. Signals were detected using
4-nitro-blue tetrazolium chloride and
5-bromo-4-chloro-3-indolyl-phosphate as substratum.
[0191] 3.1.3. Polymerisation Chain Reaction (PCR) on Placental
Genomic DNA
[0192] Placental genomic DNA was prepared according to a
conventional method (Sambrook et al., 1989). For the 5' region of
gene ICBP90, inventors used the PCR Advantage.RTM.GC genomic kit
from Clontech which is adapted to the genomic DNA regions rich in
GC. To cover the 3'-flanking regions, Taq polymerase (Sigma, St
Louis, Mo., USA) and its corresponding buffer was used. Reactions
were achieved according to the manufacturer's instructions while
using 250 ng of placental genomic DNA as matrix in a final volume
of 50 .mu.l. In order to obtain the 19 kb and 8.7 kb long intron
amplification, the PCR Expand.TM. 20kb.sup.plus system (Roche
Diagnostics, Mannheim, Germany) was used.
[0193] The reaction was completed in 100 .mu.l using 125 ng of
placental genomic DNA by reaction.
[0194] 3.1.4. Plasmidic Constructions and CAT Assays
[0195] A set of various fragments was obtained by PCR in the 5'
flanking region of gene ICBP90 using 20 nucleotide primers in order
to obtain the construction described in FIG. 10. These contain a
BamHl restriction site, and a human placental genomic DNA was used
as primer. The PCR products were digested and sub-classified
upstream from the chloramphenicol acetyl transferase (CAT) reporter
gene of a vector containing the thymidine kinase minimal promoter
(pB1CAT2). Plasmidic constructions were verified by sequencing.
COS-1 cells were cultivated in a Dulbecco milieu modified by Eagle
(DMEM) supplemented with 5% foetal calf serum. After the spreading,
the cells were transferred with the various plasmidic constructions
(5 .mu.g) using the co-precipitation technique with calcium
phosphate (Banerji et al., 1981). Analyses of CAT expression were
then carried out as describes elsewhere (Goetz et al., 1996)
[0196] 3.1.5. Chromosomal Localization of Gene ICBP90
[0197] Some metaphasic chromosomes were prepared from human
peripheral blood leukocytes according to standard protocols (Haddad
et al., 1988). Briefly, a 10 kb probe corresponding to a 5'
terminal fragment of the 16 kb clone isolated from the placental
genomic DNA screening library, was labelled with biotine-16-dUTP
(Roche Diagnostics) by "nick-translation". The probe was then
precipitated with an excess (50.times.) of Cot-1 human DNA (Life
Technologies, Rockville Md.), resuspended in 50% formamide,
1.times. SSC, pre-hybridised for 2 hours at 37.degree. C. then
hybridised overnight at 37.degree. C. The detection was carried out
using avidin-FITC (Vector Laboratories, Burlingam, Calif.).
Chromosomes were counter-stained with 4'-6-diamino-2-phenylindole
(Sigma).
[0198] 3.1.6. Northern and Western Blotting Analysis
[0199] A Northern Blotting membrane containing 2 .mu.g of polyA+
RNA by line, coming from 7 different human cancerous cell lines
(Clontech) was pre-hybridised in Express Hyb (Clontech), then
hybridised with the specific ICBP90 probe in Express Hyb at
68.degree. C. for two hours. The double-strand probe labelled with
digoxigenin was prepared from PCR amplification of a 676 bp
fragment from ICBP90 cDNA (nucleotides 806 to 1485; Genbank
accession number AF 129507) according to the manufacturer's
instructions (Roche Diagnostics).
[0200] After purification through a Micro Bio-Spin 30
chromatography column (Bio Rad, Hercules, Calif.), the specific
ICBP90 probe (5 ng/ml) was heated at 95.degree. C. for 15 minutes
then cooled on ice before addition of the hybridisation solution.
Washing after hybridisation were carried out twice in 2.times. SSC,
0.1% SD (30 minutes per wash at room temperature), then twice in
SSC 0.1 X, 0.1% SD (30 min per wash at 68.degree. C.). The membrane
was treated with solution A (0.1 M malic acid, 0.15 M NaCl at pH
7.5) then blocked by incubation with 1% blocking agent (Roche
Diagnostics) in buffer A for 30 min at room temperature.
[0201] An antibody conjugated to alcaline-phosphatase directed
against the digoxigenin (Fab fragment, Roche Diagnostics) was added
(150 mU/ml) then incubated for 30 min at room temperature. The
membrane was then washed twice with solution A, then balanced in
0.1 M tris-HCl, 0.1 M NaCl, pH 9.5. For the detection by
chemiluminescence, the inventors used agent disodium
3-(4-methoxyspiro(1,2-dioetane-3,2'-(5'-chloro)tricyclo-[3,3.1.1.sup.3,7]-
decan}-4-yl) phenyl phosphates (Roche Diagnostics) according to the
manufacturer's instructions. mRNA strips were quantified using the
NIH software Image 1.62 and expressed as a percentage of the most
abundant mRNA strip (e.g. the 5.1 kb strip of HL-60 cells).
[0202] Western Blotting analysis was carried out as describes
elsewhere (Hopfner et al., 2000). Signals were detected using
4-nitro-blue tetrazolium chloride and 5-bromo-4chloro-3-indolyl
phosphate as substrate.
[0203] 3.1.7. Local Base Alignment Research Tools, Primer
Transcription and PolyA Signal Sites Predictions
[0204] Local base alignment research tools was completed via the
National Biotechnology Information Center at the National Institute
of Health (Bethesda, Md., USA). The transcription factor library
screening with Mat Inspector software, the primer transcription
site predictions (TSS) with Neural Network, as well as the polyA
signal prediction, were all carried out at Baylor College of
Medicine (Reese et al., 1996).
[0205] 3.2. Results
[0206] 3.2.1. Isolation and Characterization of Gene ICBP90
[0207] A DNA complementary library of human placenta cloned within
the lambda GEM 12 phage was screen using a DNA probe. The screening
lead to the purification of a single positive clone with a 16 kb
insert. The sequence analysis permitted to determine that it
contained a 10 kb intronic sequence containing 3 exons (called B,
C, and D in FIG. 9A). All others screenings, namely including those
completed with PCR on BAC (Bacterial Artificial Chromosome) or YAC
(Yeast Artificial Chromosome) banks, failed to yield other positive
clones. Therefore, we decided to determine the remainder of the
gene organization directly by PCR on human placenta genomic DNA.
The biggest difficulty was to get the 5' end of the 19 kb intron.
Primers were so chosen in exon A (sense primer) and in the 5' end
of the 16 kb clone (anti-sense primer). The exon E and the 8.7 kb
intron were amplified using a sense primer in exon D and anti-sense
primer in exon F. Finally, the complete sequence of exon F up to
the poly-adenylation signal was determined using a sense primer
chosen at the beginning of exon F and the anti-sense primer in the
3' end of an EST (reference in GenBank No. AW297533) homologous to
Gene ICBP90 sequence. The complete sequence of gene ICBP90 shows
that it is made of 6 coding exons which size varies from 100 bp to
3453 bp. Most exon/intron junctions match consensus sequences for
splicing acceptor and donor sites. A poly-adenylation (AATAAA)
consensus sequence was found in the 3' region, e.g. 1152
nucleotides after the stopping codon in FIG. 9A.
[0208] 3.2.2 The 5' Region of Gene 11OEP90
[0209] The complementary DNA screening library of human thymus
cloned in lambda gt 10 phage lead to obtaining two cDNA populations
distinguishing one another from their 5' region, precisely 10 base
pairs upstream from the primer codon, i.e. in the non-translated 5'
region. These two cDNA populations predict the existence of two
alternative exons in 5' called exon I and II (FIG. 9A). We observed
that exons I and II are linked to an alternative internal splicing
site of exon A. Moreover, in a database, we found an EST (reference
in GenBank No. AI084125) corresponding to nucleotides 1290 to 1356
(FIG. 9B). The positions of these two exons and of the EST inside
the locus were determined by PCR. For that, we used primers
corresponding to the first 18 nucleotides of each exon and an
anti-sense primer selected from the first exon translated (exon A).
This strategy permitted us to rebuild the 5' region as represented
in FIGS. 9A and B, with exon I corresponding to nucleotides 1 to
134 and exon II corresponding to nucleotides 676 to 725. The EST
sequence (AI084125) is adjacent to exon A internal splicing site.
We haven't determine yet with precision the beginning of exons I,
II, and A since their sequences have been deducted from cDNA bank
screenings (FIG. 9A).
[0210] Four GC boxes (GC1 to GC4) have been found in the 5' region
(FIG. 9B). These boxes represent the potential sites of linkage for
the Sp1 transcription factor, but only one box (GC3) corresponds to
a consensus sequence, e.g. GGGGCGGGG. Besides two CCAAT boxes (CB1
and CB2) were found. Predictive analyses of sequences suggest that
two promoter regions exist in the 5' region, e.g. before the
initiation codon (ATG). Two potential transcription initiation
sites have been predicted in positions 571 and 827. The first
follows the linkage consensus sequence of Sp1 and the second
follows the GC1 box (between exons I & II, and exons II &
A, respectively). In order to determine if these two regions are
functional as promoter region, several plasmidic constructions
containing a reporter gene (the Chloramphenicol Acetyl Transferase
gene; CAT) downstream from the various potential promoters regions
were prepared. COS cells were transfected with these plasmidic
constructions. FIG. 10 shows the results obtained corresponding to
a percentage of increased basal activity. The maximal activity was
obtained with the plasmidic construction containing 1114 bp
upstream from the translation initiation site, with a 236.7%
increase of basal promoter activity (thymidine kinase gene minimal
promoter). The plasmidic construction containing 642 bp upstream
from ATG lead to a 115.6% increase whereas plasmidic construction
containing the sequence solely between exon I and II showed a
comparatively weak activity with only a 22.8% increase (FIG. 10).
These results suggest the existence of a promoter region between
exons II and A.
[0211] 3.2.3. Chromosomal Localization of Gene ICBP90
[0212] The chromosomal localization of gene ICBP90 was completed by
fluorescence in situ hybridisation (FISH). Gene ICBP90 is localized
on chromosome 19p13.3 in a telomeric region. A research carried out
at Genbank showed that a 6 Mb region in the chromosomal strip 19p
13.3 of a chromosome 19 (hybrid human/hamster 5HL2 B) specific
cosmid bank contains 147 nucleotides coding for ICBP90 amino acids
746 to 793. This sequence has been localized between the STS
(sequence tagged site) markers D 19S883 and D 19S325.
[0213] 3.2.4 ICBP90 Expression in Various Cellular Lineages
[0214] ICBP90 participates in the regulation of the gene
TopII.alpha. expression (Hopfner et al., 2000). As TopII.alpha. is
expressed 3rd differential manner in various tumours and cellular
lineages, ICBP90 itself is susceptible to have a complex regulation
in term of activity and genic expression.
[0215] In a first step towards understanding the mechanisms
regulating gene ICBP90 expression, ICBP90 mRNA was analysed in
various cellular lineages. ICBP90 mRNA was studied in the HL60
cellular lineage derived from promyelocytic leukaemia (lineage 1),
Hela S3 cells (lineage 2), MOLT-4 lymphoblastic leukaemia cells,
Raji Burkitt lymphoma cells (lineage 5), SW 480 colorectal
adenocarcinoma (lineage 6), A549 lung carcinoma cells (lineage 7)
(FIG. 11A).
[0216] Two 4.3 and 5.1 kb bands of mRNA are observed. The relative
amounts of mRNA in the bands vary according to the cell type. The
histogram in FIG. 11A shows the levels of mRNA in the hands of each
of the cell lines, expressed in percentage of the maximum amount of
5.1 kb bands of mRNA observed in the HL-60 cells (line 1, FIG.
11A). In the MOLT-4 cells, only the 4.3 kb band of mRNA is
observed, while in the cells from promyelocytic leukaemia the 5.1
kb band is predominant. In the Raji cells of Burkitt's lymphoma,
only the 5.1 kb band is detected. Approximately equal amounts of
the two types of mRNA are observed in the other cell lines, that
is, the Hela, K562, A549, SW580 cells. For the HL-60 cells,
nevertheless, the 5.1 kb mRNA is more strongly expressed than the
4.3 kb mRNA. Other analyses have been undertaken on the Hela cells
to confirm that the 2 transcripts originate from the transcription
of the ICBP90 gene. A cDNA probe of 626 bp labelled with
digoxigenin localized immediately upstream of the poly A signal
(that is, the exon F) and used as probe for Northern Blotting
experiments, has produced the same results, that is, the appearance
of two 4.3 kb and 5.1 kb bands of mRNA. This result confirms that
the two forms of mRNA are generated from a single gene.
[0217] The inventors have also studied the expression of the ICBP90
protein in order to determine if these two isoforms of mRNA are
likely to code for two different proteins.
[0218] FIG. 11B shows the expression profile of ICBP90 in protein
extracts of MOLT-4 and Hela cells. While a single band of 97 kDa is
observed in the MOLT-4 cells, in the Hela cells, beside the 97-kDa
band that is doubled, several other bands with a lower molecular
weight are observed. These results suggest that in the MOLT-4
cells, an mRNA codes for a single form of ICBP90. Conversely, in
the Hela cells, the two mRNA are likely to lead to the production
of different isoforms of ICBP90.
[0219] 3.3 Comments
[0220] The ICBP90 gene is spread over approximately 35.8 kb. Six
translated exons and two untranslated exons, and then, seven
introns have been identified by the inventors. The two zinc-finger
domains of ICBP90 are coded by the same exon (exon F) in contrast
to the receptor gene for human estrogens in which each of the
presumed zinc fingers of the DNA binding domain of the receptor are
coded separately (Ponglikitmongkol et al. (1988)). The
"ubiquitin-like" domain of ICBP90 is coded by exons A and B while
the "leucine zipper" is coded by exon B. Interestingly, only exon F
is likely to code for a functional protein because it codes for two
nuclear localization signals, the zinc-finger domains and several
presumed sites of phosphorylation. Two large 8.7 kb and 19 kb
introns have been found.
[0221] The ICBP90 gene has been localized in the chromosome region
19p13.3. Several other genes have been localized in this region,
for example the Nuclear Factor I/C (also a CCAAT binding
transcription factor) (Qain et al. (1995)). Interestingly, an
atypical translocation t(7;19) in the acute myelomonocytic
leukaemia, involving a fragile site at the 19p13.3 locus has been
described (Sherer et al. (1991)). Also, it has been suggested that
the genes involved in the development of pancreatic carcinomas are
localized at 19p13.3 and 19q13.1-13.2 (Hoglund et al. (1998)).
Rearrangements of the 14q32.3 and 19p13.3 bands with a preferential
deletion of the short arm of chromosome 1 form non-random
chromosome alterations in multiple myeloma and leukaemia of cells
of the plasma (Taniwaki et al. (1996)). Other genes have been
localized in this region; they include a gene involved in
adenocarcinoma of the Peutz-Jeghers syndrome (Gruba et al. (1998)).
Also, it has been suggested that the presumed tumour suppressor
gene for malignant adenoma is localized on D19S216 at the 19p13.3
chromosome band that plays an important role in tumourigenesis of
malignant adenoma (Lee et al. (1998)).
[0222] The analysis of the sequence of the 5' region of the ICBP90
gene has revealed the existence of several untranslated exons with
a promoter region between exons II and A and probably a second
weaker promoter localized between exons I and II. The promoter
region between exons II and A is a promoter without TATA sequence
suggesting that the ICBP90 gene may be a housekeeping gene at least
when this promoter is involved. In this sense, it strongly
resembles promoter regions of the genes ATF.alpha. (Goetz et al.,
1996), CRE-BP1/ATF2 (Nagase et al., 1990) and TopII.alpha.
(Hochhauser et al., 1992) which do not contain canonical TATA boxes
but several SP-1 binding sites.
[0223] The GC and/or CCAAT boxes are likely to be involved in the
regulation of the expression of the ICBP90 gene via transcription
factors SP-1 and the CCAAT binding proteins. Furthermore, given
that the ICBP90 protein is a CCAAT binding protein, ICBP90 is also
likely to regulate its own expression.
[0224] A data library of transcription factors has been screened
with the aid of the Mat Inspector computer program from the Baylor
College of Medicine and numerous binding sites of transcription
factors have been identified in the sequence preceding the ATG
codon (FIG. 9B).Among these binding sites for the transcription
factors it is interesting to note binding sites of the AP-2
transcription factor regulated during the development and which
controls the DR-nm23 gene expression (Martinez et al. (1997)), the
binding sites of the "zinc-finger" myeloid protein MZF 1 which is
involved in the regulation of hematopoiesis (Hromas et al.
(1996)).
[0225] The Northern Blotting analysis has demonstrated that two
populations of mRNA exist, 4.3 kb and 5.1 kb. Interestingly, each
population presents a cellular specificity. For example, the
lymphoblast cells MOLT-4 only express the 4.3 kb mRNA, while in the
Raji cells of Burkitt's lymphoma (mature B lymphocytes), only the
5.1 kb transcript is observed. The HL-60 cells express more 5.1 kb
mRNA then 4.3 kb mRNA. The HL-60 cells and the Raji cells of
Burkitt's lymphoma are more differentiated than the MOLT-4 cells
suggesting that the levels of expression of the 5.1 kb transcript
relative to that of 4.3 kb may be directly correlated with the
state of differentiation of the cells.
[0226] Interestingly, an expressed sequence tag (EST, Expressed
Sequence Tag) corresponding to the 5' sequence of the exon A has
been identified from anaplastic oligodendroglioma (Genbank
Accession No. AI 084 125) while an EST corresponding to the
inclusion of exon II has been isolated from a mixture of tumours
with germinal cells (Genbank Accession No. AI 968 662). The results
of the inventors therefore suggest that the regulation of the
ICBP90 transcripts is comparable to that which happens with the
oestrogen receptor. In fact, six different transcripts coding a
common protein, but differing in the untranslated 5' region because
of an alternative splicing of upstream exons, have been reported
(Flouriot et al., 1998 and Grandien, 1996).
[0227] The Western Blotting analysis shows a major band at 97 kDa
in the MOLT-4 cells while several bands are observed in the Hela
cells (Figure llB). This data is in agreement with the existence of
several ICBP90 mRNA and/or isoforms of the ICBP90 protein for which
the level of expression may be controlled in a cell-specific
manner.
[0228] Two protein isoforms for the oestrogen receptor have been
described (Griffin et al., 1999) which differ from each other by
the 41 N-terminal amino acids. The 97 kDa double band observed from
the Hela cells (FIG. 11B) is therefore likely to represent two
isoforms differing by their N-terminal end. To do this, the exon A
coding for 47 amino acids is spliced outside the reading frame, and
consequently, the protein-coding region begins with exon B.
Nevertheless, it is also possible that there are other exons likely
to be transcribed in other tissues.
[0229] Also, the 8.7 kb intron (that is, between exon D and E) is
likely to contain a promoter region which may lead to ICBP90
isoforms with lower molecular weight than those observed in the
Hela cells in proliferation (FIG. 11B). Interestingly, the tissue
specificity of the different mRNA of the oestrogen receptor is
determined by different promoters for which the activity appears to
be altered in the cell lines of breast cancer (Flouriot et al.,
1998).
[0230] All these results suggest that the ICBP90 gene and the
ICBP90 protein present characteristics common with members of the
family of the receptor for retinoic acid, steroids, thyroid
hormones where it concerns gene and protein structures.
[0231] In fact, the inventors have demonstrated experimentally, by
using the double-hybrid technique, the existence of interactions
between the ICBP90 protein and TIP60 (Tat Interactive Protein, 60
kDa). The TIP60 protein has very recently been described as being a
coactivator of the nuclear receptor, especially the receptor for
the androgens (Brady M E et al., 1999).
[0232] Because of this, ICBP90 is capable of playing the role of a
nuclear receptor on which an endogenous ligand is bound. Therefore,
it is also within the scope of the present invention to use the
ICBP90 polypeptide of the invention to isolate, screen, and
identify the endogenous ligand. It is also within the scope of the
invention to use the ICB90 polypeptide of the invention to isolate,
screen identify natural or synthetic, biological or chemical,
agonist or antagonist molecules of this natural ligand.
REFERENCES
[0233] Austin et al. (1993), Biochem. Biophys. Acta, 1172,
283-291
[0234] Banerji, J. et al. (1981), Cell, 27: 299-308.
[0235] Barany, F. (1991), Proc. Natl. Acad. Sci. USA, 88,
189-193.
[0236] Boritzki, T. J. et al. (1988), Biochem. Pharmacol., 37,
4063-4068.
[0237] Brady, M. E. et al. (1999), J. Biol Chem., 274:
17599-17604.
[0238] Brandt, T. L. et al. (1997), J. Biol. Chem., 272,
6278-6284.
[0239] Brou, C. et al. (1993), EMBO J., 12, 489-499.
[0240] Buckholz, R. G. (1993), Curr. Op. Biotechnology, 4,
538-542.
[0241] Burg, J. L. et al. (1996), Mol. and Cell. Probes, 10,
257-271.
[0242] Chu, B. C. F. et al. (1986), Nucleic Acids Res., 14,
5591-5603.
[0243] Chung, T. D. Y. et al. (1989), Proc. Natl. Acad. Sci. USA,
86, 9431-9435.
[0244] Darzynkiewicz et al. (1994), Methods in Cell Biology, 41,
421-435.
[0245] Deffie, A. M. et al. (1989), Cancer Res., 49, 58-62.
[0246] DeVries, L. et al. (1995), Proc. Natl. Acad. Sci. USA, 92,
11916-11920.
[0247] Devys et al. (1993), Nature Genet., 4, 335-340.
[0248] Drake, F. H. et al., Biochemistry, 28, 8154-8160.
[0249] Duck, P. et al. (1990), Biotechniques, 9, 142-147.
[0250] Edwards, C. P. and Aruffo, A. (1993), Curr. Op.
Biotechnology, 4, 558-563.
[0251] Erlich, H. A. (1989), New York: Stockton Press.
[0252] Flouriot et al. (1998), Mol. Endocrinol., 12:1239-254.
[0253] Fry, A. M. et al. (1991), Cancer Res., 51, 6592-6595.
[0254] Furth et al. (1992), Anal. Biochem., 205: 365.
[0255] Gaub, M. P. et al. (1998). J. Histochem. Cytochem., 46,
1103-1111.
[0256] Goetz, J. et al. (1996), J. Biol. Chem.,
271:29589-29598.
[0257] Goswami, P. C. et al. (1996), Mol. Cell. Biol., 16,
1500-1508.
[0258] Grandien (1996), Mol. Cell. Endocrinol., 116: 207-212.
[0259] Griffin et al. (1999), Mol. Endocrinol., 13: 1571-1587.
[0260] Gruba et al. (1998), Cancer Res., 58: 5267-5270.
[0261] Guatelli, J. C. et al. (1990), Proc. Natl. Acad. Sci. USA,
87, 1874-1878.
[0262] Guinee, D. G. et al. (1996), Cancer, 78, 729-735.
[0263] Haddad et al. (1988), Human Genet. 103: 619-625.
[0264] Heck, M. M. et al. (1988), Proc. Natl. Acad. Sci. USA, 85,
1086-1090.
[0265] Herzog, C. E. and Zwelling, L. A. (1997), Biochem. Biophys.
Res. Commun., 232, 608-612.
[0266] Hochhauser, D. et al. (1992), J. Biol. Chem., 267,
18961-18965.
[0267] Hoglund et al. (1998), Genes Chromosomes Cancer 21:8-16.
[0268] Hopfner et al. (2000), Cancer Res., 60:121-128.
[0269] Hromas et al. (1996), Curr. Top. Microbiol. Chem.,
211:159-164.
[0270] Innis, M. A. et al. (1990), Academic Press.
[0271] Inouye, C. et al. (1994), DNA Cell Biol., 13, 731-742.
[0272] Isaacs, R. J. et al. Biochem. Biophys. Acta, 1400,
121-137.
[0273] Isaacs, R. J. et al. (1996), J. Biol. Chem., 271,
16741-16747.
[0274] Jenkins, J. R. et al. (1992), Nucleic Acids Res., 20,
5587-5592.
[0275] Kassel, O. et al. (1998), Mol. Pharmacol., 54,
1073-1079.
[0276] Kennerdell, J. R. and Carthew, R. W. (1998), Cell, 95,
1017-1026.
[0277] Kievitis, T. et al. (1975), J. Virol. Methods, 35,
273-286.
[0278] Kohler, G. et al. (1975), Nature, 256 (5517), 495-497.
[0279] Kubo, T. et al., (1995), Cancer Res., 55, 3860-3864.
[0280] Kwoh, D. Y. et al., (1989), Proc. Natl. Acad. Sci. USA, 86,
1173-1177.
[0281] Landegren, U. et al. (1988), Science, 241, 1077-1080.
[0282] Lavie, J. et al. (1999), J. Biol. Chem., 274, 2308-2314.
[0283] Lee et al. (1998), Cancer Res., 58: 1140-1143.
[0284] Lim, K. et al. (1998), Biochem. Mol. Biol. Int., 46,
35-42.
[0285] Lizardi, P. M. et al. (1988), Bio/technology, 6,
1197-1202.
[0286] Lucknow, V. A. (1993), Curr. Op. Biotechnology, 4,
564-572.
[0287] Martinez, et al. (1997), Cancer Res., 57: 1180-1187.
[0288] Matthews, J. A. et al. (1988), Anal. Biochem., 169:1-25.
[0289] Miele, E. A. et al. (1983), J. Mol. Biol., 171, 281-295.
[0290] Nagase et al. (1990), J. Biol. Chem., 265:17300-17305.
[0291] Nitiss, J. L. (1998), Biochem. Biophys. Acta, 1400,
63-81.
[0292] Olins, P. O. and Lee, S. C. (1993), Curr. Op. Biotechnology,
4, 520-525.
[0293] Pommier, Y. et al. (1994), Cancer Invest., 12, 530-542.
[0294] Ponglikitmongkoi et al. (1988), EMBO J. 7:3385-3388.
[0295] Rio, M. C. et al. (1987), Proc. Natl. Acad. Sci. USA, 84,
9243-9247.
[0296] Qian et al. (1995), Genomics, 28:66-73.
[0297] Reese et al. (1996), Large Scale Sequencing Specific Neural
Networks for Promoter and Splice Recognition. Biocomputing:
Proceedings of the 1995 Pacific Symposium. Edited by Lawrence
Hunter and Terri E. Wood Scientific Singapore, 1996, Jan. 27,
1996.
[0298] Rochette-Egly, C. et al. (1997), Cell, 90, 97-107.
[0299] Roskell, D. E. and Biddolph, S. C. (1999), Eur. J. Med. Res.
26, 105-106.
[0300] Rolfs, A. et al. (1991), Berlin: Springer-Verlag.
[0301] Sambrook, J. et al. (1989), Molecular Cloning: A Laboratory
Manual, Sec. Ed. Cold Spring Harbor Lab., Cold Spring Harbor,
N.Y.
[0302] Sandri, M.I. et al. (1996), Nucleic Acids Res., 24,
4464-4470.
[0303] Segev, D. (1992), Kessler C. Springer Verlag, Berlin, New
York, 197-205.
[0304] Sherer et al. (1991), Cancer Genet. Cytogenet., 57:
169-173.
[0305] Stone, B. B. et al. (1996) Mol. and Cell. Probes,
10:359-370.
[0306] Tang et al. (1992), Nature, 356:152.
[0307] Taniwaki et al. (1996), Leuk. Lymphoma, 21:25-30.
[0308] Tsai-plugfelder, M. et al. (1988), Proc. Natl. Acad. Sci.
USA, 85, 7177-7181.
[0309] Walker, G. T. et al. (1992), Nucleic Acids Res.,
20:1691-1696.
[0310] Wang, J. C. (1996), Ann. Rev. Biochem., 65, 635-692.
[0311] Wang, M. M. and Reed, R. R. (1993), Nature (London), 364,
121-126.
[0312] Yamazaki et al. (1996), Acta Oncol., 35, 417-423.
Sequence CWU 1
1
24 1 2382 DNA Homo sapiens CDS (1)..(2379) 1 atg tgg atc cag gtt
cgg acc atg gat ggg agg cag acc cac acg gtg 48 Met Trp Ile Gln Val
Arg Thr Met Asp Gly Arg Gln Thr His Thr Val 1 5 10 15 gac tcg ctg
tcc agg ctg acc aag gtg gag gag ctg agg cgg aag atc 96 Asp Ser Leu
Ser Arg Leu Thr Lys Val Glu Glu Leu Arg Arg Lys Ile 20 25 30 cag
gag ctg ttc cac gtg gag cca ggc ctg cag agg ctg ttc tac agg 144 Gln
Glu Leu Phe His Val Glu Pro Gly Leu Gln Arg Leu Phe Tyr Arg 35 40
45 ggc aaa cag atg gag gac ggc cat acc ctc ttc gac tac gag gtc cgc
192 Gly Lys Gln Met Glu Asp Gly His Thr Leu Phe Asp Tyr Glu Val Arg
50 55 60 ctg aat gac acc atc cag ctc ctg gtc cgc cag agc ctc gtg
ctc ccc 240 Leu Asn Asp Thr Ile Gln Leu Leu Val Arg Gln Ser Leu Val
Leu Pro 65 70 75 80 cac agc acc aag gag cgg gac tcc gag ctc tcc gac
acc gac tcc ggc 288 His Ser Thr Lys Glu Arg Asp Ser Glu Leu Ser Asp
Thr Asp Ser Gly 85 90 95 tgc tgc ctg ggc cag agt gag tca gac aag
tcc tcc acc cac ggt gag 336 Cys Cys Leu Gly Gln Ser Glu Ser Asp Lys
Ser Ser Thr His Gly Glu 100 105 110 gcg gcc gcc gag act gac agc agg
cca gcc gat gag gac atg tgg gat 384 Ala Ala Ala Glu Thr Asp Ser Arg
Pro Ala Asp Glu Asp Met Trp Asp 115 120 125 gag acg gaa ttg ggg ctg
tac aag gtc aat gag tac gtc gat gct cgg 432 Glu Thr Glu Leu Gly Leu
Tyr Lys Val Asn Glu Tyr Val Asp Ala Arg 130 135 140 gac acg aac atg
ggg gcg tgg ttt gag gcg cag gtg gtc agg gtg acg 480 Asp Thr Asn Met
Gly Ala Trp Phe Glu Ala Gln Val Val Arg Val Thr 145 150 155 160 cgg
aag gcc ccc tcc cgg gac gag ccc tgc agc tcc acg tcc agg ccg 528 Arg
Lys Ala Pro Ser Arg Asp Glu Pro Cys Ser Ser Thr Ser Arg Pro 165 170
175 gcg ctg gag gag gac gtc att tac cac gtg aaa tac gac gac tac ccg
576 Ala Leu Glu Glu Asp Val Ile Tyr His Val Lys Tyr Asp Asp Tyr Pro
180 185 190 gag aac ggc gtg gtc cag atg aac tcc agg gac gtc cga gcg
cgc gcc 624 Glu Asn Gly Val Val Gln Met Asn Ser Arg Asp Val Arg Ala
Arg Ala 195 200 205 cgc acc atc atc aag tgg cag gac ctg gag gtg ggc
cag gtg gtc atg 672 Arg Thr Ile Ile Lys Trp Gln Asp Leu Glu Val Gly
Gln Val Val Met 210 215 220 ctc aac tac aac ccc gac aac ccc aag gag
cgg ggc ttc tgg tac gac 720 Leu Asn Tyr Asn Pro Asp Asn Pro Lys Glu
Arg Gly Phe Trp Tyr Asp 225 230 235 240 gcg gag atc tcc agg aag cgc
gag acc agg acg gcg cgg gaa ctc tac 768 Ala Glu Ile Ser Arg Lys Arg
Glu Thr Arg Thr Ala Arg Glu Leu Tyr 245 250 255 gcc aac gtg gtg ctg
ggg gat gat tct ctg aac gac tgt cgg atc atc 816 Ala Asn Val Val Leu
Gly Asp Asp Ser Leu Asn Asp Cys Arg Ile Ile 260 265 270 ttc gtg gac
gaa gtc ttc aag att gag cgg ccg ggt gaa ggg agc ccc 864 Phe Val Asp
Glu Val Phe Lys Ile Glu Arg Pro Gly Glu Gly Ser Pro 275 280 285 atg
gtt gac aac ccc atg aga cgg aag agc ggg ccg tcc tgc aag cac 912 Met
Val Asp Asn Pro Met Arg Arg Lys Ser Gly Pro Ser Cys Lys His 290 295
300 tgc aag gac gac gtg aac aga ctc tgc agg gtc tgc gcc tgc cac ctg
960 Cys Lys Asp Asp Val Asn Arg Leu Cys Arg Val Cys Ala Cys His Leu
305 310 315 320 tgc ggg ggc cgg cag gac ccc gac aag cag ctc atg tgc
gat gag tgc 1008 Cys Gly Gly Arg Gln Asp Pro Asp Lys Gln Leu Met
Cys Asp Glu Cys 325 330 335 gac atg gcc ttc cac atc tac tgc ctg gac
ccg ccc ctc agc agt gtt 1056 Asp Met Ala Phe His Ile Tyr Cys Leu
Asp Pro Pro Leu Ser Ser Val 340 345 350 ccc agc gag gac gag tgg tac
tgc cct gag tgc cgg aat gat gcc agc 1104 Pro Ser Glu Asp Glu Trp
Tyr Cys Pro Glu Cys Arg Asn Asp Ala Ser 355 360 365 gag gtg gta ctg
gcg gga gag cgg ctg aga gag agc aag aag aat gcg 1152 Glu Val Val
Leu Ala Gly Glu Arg Leu Arg Glu Ser Lys Lys Asn Ala 370 375 380 aag
atg gcc tcg gcc aca tcg tcc tca cag cgg gac tgg ggc aag ggc 1200
Lys Met Ala Ser Ala Thr Ser Ser Ser Gln Arg Asp Trp Gly Lys Gly 385
390 395 400 atg gcc tgt gtg ggc cgc acc aag gaa tgt acc atc gtc ccg
tcc aac 1248 Met Ala Cys Val Gly Arg Thr Lys Glu Cys Thr Ile Val
Pro Ser Asn 405 410 415 cac tac gga ccc atc ccg ggg atc ccc gtg ggc
acc atg tgg cgg ttc 1296 His Tyr Gly Pro Ile Pro Gly Ile Pro Val
Gly Thr Met Trp Arg Phe 420 425 430 cga gtc cag gtc agc gag tcg ggt
gtc cat cgg ccc cac gtg gct ggc 1344 Arg Val Gln Val Ser Glu Ser
Gly Val His Arg Pro His Val Ala Gly 435 440 445 atc cat ggc cgg agc
aac gac gga tcg tac tcc cta gtc ctg gcg ggg 1392 Ile His Gly Arg
Ser Asn Asp Gly Ser Tyr Ser Leu Val Leu Ala Gly 450 455 460 ggc tat
gag gat gat gtg gac cat ggg aat ttt ttc aca tac acg ggt 1440 Gly
Tyr Glu Asp Asp Val Asp His Gly Asn Phe Phe Thr Tyr Thr Gly 465 470
475 480 agt ggt ggt cga gat ctt tcc ggc aac aag agg acc gcg gaa cag
tct 1488 Ser Gly Gly Arg Asp Leu Ser Gly Asn Lys Arg Thr Ala Glu
Gln Ser 485 490 495 tgt gat cag aaa ctc acc aac acc aac agg gcg ctg
gct ctc aac tgc 1536 Cys Asp Gln Lys Leu Thr Asn Thr Asn Arg Ala
Leu Ala Leu Asn Cys 500 505 510 ttt gct ccc atc aat gac caa gaa ggg
gcc gag gcc aag gac tgg cgg 1584 Phe Ala Pro Ile Asn Asp Gln Glu
Gly Ala Glu Ala Lys Asp Trp Arg 515 520 525 tcg ggg aag ccg gtc agg
gtg gtg cgc aat gtc aag ggt ggc aag aat 1632 Ser Gly Lys Pro Val
Arg Val Val Arg Asn Val Lys Gly Gly Lys Asn 530 535 540 agc aag tac
gcc ccc gct gag ggc aac cgc tac gat ggc atc tac aag 1680 Ser Lys
Tyr Ala Pro Ala Glu Gly Asn Arg Tyr Asp Gly Ile Tyr Lys 545 550 555
560 gtt gtg aaa tac tgg ccc gag aag ggg aag tcc ggg ttt ctc gtg tgg
1728 Val Val Lys Tyr Trp Pro Glu Lys Gly Lys Ser Gly Phe Leu Val
Trp 565 570 575 cgc tac ctt ctg cgg agg gac gat gat gag cct ggc cct
tgg acg aag 1776 Arg Tyr Leu Leu Arg Arg Asp Asp Asp Glu Pro Gly
Pro Trp Thr Lys 580 585 590 gag ggg aag gac cgg atc aag aag ctg ggg
ctg acc atg cag tat cca 1824 Glu Gly Lys Asp Arg Ile Lys Lys Leu
Gly Leu Thr Met Gln Tyr Pro 595 600 605 gaa ggc tac ctg gaa gcc ctg
gcc aac cga gag cga gag aag gag aac 1872 Glu Gly Tyr Leu Glu Ala
Leu Ala Asn Arg Glu Arg Glu Lys Glu Asn 610 615 620 agc aag agg gag
gag gag gag cag cag gag ggg ggc ttc gcg tcc ccc 1920 Ser Lys Arg
Glu Glu Glu Glu Gln Gln Glu Gly Gly Phe Ala Ser Pro 625 630 635 640
agg acg ggc aag ggc aag tgg aag cgg aag tcg gca gga ggt ggc ccg
1968 Arg Thr Gly Lys Gly Lys Trp Lys Arg Lys Ser Ala Gly Gly Gly
Pro 645 650 655 agc agg gcc ggg tcc ccg cgc cgg aca tcc aag aaa acc
aag gtg gag 2016 Ser Arg Ala Gly Ser Pro Arg Arg Thr Ser Lys Lys
Thr Lys Val Glu 660 665 670 ccc tac agt ctc acg gcc cag cag agc agc
ctc atc aga gag gac aag 2064 Pro Tyr Ser Leu Thr Ala Gln Gln Ser
Ser Leu Ile Arg Glu Asp Lys 675 680 685 agc aac gcc aag ctg tgg aat
gag gtc ctg gcg tca ctc aag gac cgg 2112 Ser Asn Ala Lys Leu Trp
Asn Glu Val Leu Ala Ser Leu Lys Asp Arg 690 695 700 ccg gcg agc ggc
agc ccg ttc cag ttg ttc ctg agt aaa gtg gag gag 2160 Pro Ala Ser
Gly Ser Pro Phe Gln Leu Phe Leu Ser Lys Val Glu Glu 705 710 715 720
acg ttc cag tgt atc tgc tgt cag gag ctg gtg ttc cgg ccc atc acg
2208 Thr Phe Gln Cys Ile Cys Cys Gln Glu Leu Val Phe Arg Pro Ile
Thr 725 730 735 acc gtg tgc cag cac aac gtg tgc aag gac tgc ctg gac
aga tcc ttt 2256 Thr Val Cys Gln His Asn Val Cys Lys Asp Cys Leu
Asp Arg Ser Phe 740 745 750 cgg gca cag gtg ttc agc tgc cct gcc tgc
cgc tac gac ctg ggc cgc 2304 Arg Ala Gln Val Phe Ser Cys Pro Ala
Cys Arg Tyr Asp Leu Gly Arg 755 760 765 agc tat gcc atg cag gtg aac
cag cct ctg cag acc gtc ctc aac cag 2352 Ser Tyr Ala Met Gln Val
Asn Gln Pro Leu Gln Thr Val Leu Asn Gln 770 775 780 ctc ttc ccc ggc
tac ggc aat ggc cgg tga 2382 Leu Phe Pro Gly Tyr Gly Asn Gly Arg
785 790 2 793 PRT Homo sapiens 2 Met Trp Ile Gln Val Arg Thr Met
Asp Gly Arg Gln Thr His Thr Val 1 5 10 15 Asp Ser Leu Ser Arg Leu
Thr Lys Val Glu Glu Leu Arg Arg Lys Ile 20 25 30 Gln Glu Leu Phe
His Val Glu Pro Gly Leu Gln Arg Leu Phe Tyr Arg 35 40 45 Gly Lys
Gln Met Glu Asp Gly His Thr Leu Phe Asp Tyr Glu Val Arg 50 55 60
Leu Asn Asp Thr Ile Gln Leu Leu Val Arg Gln Ser Leu Val Leu Pro 65
70 75 80 His Ser Thr Lys Glu Arg Asp Ser Glu Leu Ser Asp Thr Asp
Ser Gly 85 90 95 Cys Cys Leu Gly Gln Ser Glu Ser Asp Lys Ser Ser
Thr His Gly Glu 100 105 110 Ala Ala Ala Glu Thr Asp Ser Arg Pro Ala
Asp Glu Asp Met Trp Asp 115 120 125 Glu Thr Glu Leu Gly Leu Tyr Lys
Val Asn Glu Tyr Val Asp Ala Arg 130 135 140 Asp Thr Asn Met Gly Ala
Trp Phe Glu Ala Gln Val Val Arg Val Thr 145 150 155 160 Arg Lys Ala
Pro Ser Arg Asp Glu Pro Cys Ser Ser Thr Ser Arg Pro 165 170 175 Ala
Leu Glu Glu Asp Val Ile Tyr His Val Lys Tyr Asp Asp Tyr Pro 180 185
190 Glu Asn Gly Val Val Gln Met Asn Ser Arg Asp Val Arg Ala Arg Ala
195 200 205 Arg Thr Ile Ile Lys Trp Gln Asp Leu Glu Val Gly Gln Val
Val Met 210 215 220 Leu Asn Tyr Asn Pro Asp Asn Pro Lys Glu Arg Gly
Phe Trp Tyr Asp 225 230 235 240 Ala Glu Ile Ser Arg Lys Arg Glu Thr
Arg Thr Ala Arg Glu Leu Tyr 245 250 255 Ala Asn Val Val Leu Gly Asp
Asp Ser Leu Asn Asp Cys Arg Ile Ile 260 265 270 Phe Val Asp Glu Val
Phe Lys Ile Glu Arg Pro Gly Glu Gly Ser Pro 275 280 285 Met Val Asp
Asn Pro Met Arg Arg Lys Ser Gly Pro Ser Cys Lys His 290 295 300 Cys
Lys Asp Asp Val Asn Arg Leu Cys Arg Val Cys Ala Cys His Leu 305 310
315 320 Cys Gly Gly Arg Gln Asp Pro Asp Lys Gln Leu Met Cys Asp Glu
Cys 325 330 335 Asp Met Ala Phe His Ile Tyr Cys Leu Asp Pro Pro Leu
Ser Ser Val 340 345 350 Pro Ser Glu Asp Glu Trp Tyr Cys Pro Glu Cys
Arg Asn Asp Ala Ser 355 360 365 Glu Val Val Leu Ala Gly Glu Arg Leu
Arg Glu Ser Lys Lys Asn Ala 370 375 380 Lys Met Ala Ser Ala Thr Ser
Ser Ser Gln Arg Asp Trp Gly Lys Gly 385 390 395 400 Met Ala Cys Val
Gly Arg Thr Lys Glu Cys Thr Ile Val Pro Ser Asn 405 410 415 His Tyr
Gly Pro Ile Pro Gly Ile Pro Val Gly Thr Met Trp Arg Phe 420 425 430
Arg Val Gln Val Ser Glu Ser Gly Val His Arg Pro His Val Ala Gly 435
440 445 Ile His Gly Arg Ser Asn Asp Gly Ser Tyr Ser Leu Val Leu Ala
Gly 450 455 460 Gly Tyr Glu Asp Asp Val Asp His Gly Asn Phe Phe Thr
Tyr Thr Gly 465 470 475 480 Ser Gly Gly Arg Asp Leu Ser Gly Asn Lys
Arg Thr Ala Glu Gln Ser 485 490 495 Cys Asp Gln Lys Leu Thr Asn Thr
Asn Arg Ala Leu Ala Leu Asn Cys 500 505 510 Phe Ala Pro Ile Asn Asp
Gln Glu Gly Ala Glu Ala Lys Asp Trp Arg 515 520 525 Ser Gly Lys Pro
Val Arg Val Val Arg Asn Val Lys Gly Gly Lys Asn 530 535 540 Ser Lys
Tyr Ala Pro Ala Glu Gly Asn Arg Tyr Asp Gly Ile Tyr Lys 545 550 555
560 Val Val Lys Tyr Trp Pro Glu Lys Gly Lys Ser Gly Phe Leu Val Trp
565 570 575 Arg Tyr Leu Leu Arg Arg Asp Asp Asp Glu Pro Gly Pro Trp
Thr Lys 580 585 590 Glu Gly Lys Asp Arg Ile Lys Lys Leu Gly Leu Thr
Met Gln Tyr Pro 595 600 605 Glu Gly Tyr Leu Glu Ala Leu Ala Asn Arg
Glu Arg Glu Lys Glu Asn 610 615 620 Ser Lys Arg Glu Glu Glu Glu Gln
Gln Glu Gly Gly Phe Ala Ser Pro 625 630 635 640 Arg Thr Gly Lys Gly
Lys Trp Lys Arg Lys Ser Ala Gly Gly Gly Pro 645 650 655 Ser Arg Ala
Gly Ser Pro Arg Arg Thr Ser Lys Lys Thr Lys Val Glu 660 665 670 Pro
Tyr Ser Leu Thr Ala Gln Gln Ser Ser Leu Ile Arg Glu Asp Lys 675 680
685 Ser Asn Ala Lys Leu Trp Asn Glu Val Leu Ala Ser Leu Lys Asp Arg
690 695 700 Pro Ala Ser Gly Ser Pro Phe Gln Leu Phe Leu Ser Lys Val
Glu Glu 705 710 715 720 Thr Phe Gln Cys Ile Cys Cys Gln Glu Leu Val
Phe Arg Pro Ile Thr 725 730 735 Thr Val Cys Gln His Asn Val Cys Lys
Asp Cys Leu Asp Arg Ser Phe 740 745 750 Arg Ala Gln Val Phe Ser Cys
Pro Ala Cys Arg Tyr Asp Leu Gly Arg 755 760 765 Ser Tyr Ala Met Gln
Val Asn Gln Pro Leu Gln Thr Val Leu Asn Gln 770 775 780 Leu Phe Pro
Gly Tyr Gly Asn Gly Arg 785 790 3 45 DNA Homo sapiens CDS (1)..(45)
3 acc cac ggt gag gcg gcc gcc gag act gac agc agg cca gcc gat 45
Thr His Gly Glu Ala Ala Ala Glu Thr Asp Ser Arg Pro Ala Asp 1 5 10
15 4 15 PRT Homo sapiens 4 Thr His Gly Glu Ala Ala Ala Glu Thr Asp
Ser Arg Pro Ala Asp 1 5 10 15 5 78 DNA Homo sapiens CDS (1)..(78) 5
atg gtt gac aac ccc atg aga cgg aag agc ggg ccg tcc tgc aag cac 48
Met Val Asp Asn Pro Met Arg Arg Lys Ser Gly Pro Ser Cys Lys His 1 5
10 15 tgc aag gac gac gtg aac aga ctc tgc agc 78 Cys Lys Asp Asp
Val Asn Arg Leu Cys Ser 20 25 6 26 PRT Homo sapiens 6 Met Val Asp
Asn Pro Met Arg Arg Lys Ser Gly Pro Ser Cys Lys His 1 5 10 15 Cys
Lys Asp Asp Val Asn Arg Leu Cys Ser 20 25 7 525 DNA Homo sapiens
CDS (1)..(522) 7 cga gag aag gag aac agc aag agg gag gag gag gag
cag cag gag ggg 48 Arg Glu Lys Glu Asn Ser Lys Arg Glu Glu Glu Glu
Gln Gln Glu Gly 1 5 10 15 ggc ttc gcg tcc ccc agg acg ggc aag ggc
aag tgg aag cgg aag tcg 96 Gly Phe Ala Ser Pro Arg Thr Gly Lys Gly
Lys Trp Lys Arg Lys Ser 20 25 30 gca gga ggt ggc ccg agc agg gcc
ggg tcc ccg cgc cgg aca tcc aag 144 Ala Gly Gly Gly Pro Ser Arg Ala
Gly Ser Pro Arg Arg Thr Ser Lys 35 40 45 aaa acc aag gtg gag ccc
tac agt ctc acg gcc cag cag agc agc ctc 192 Lys Thr Lys Val Glu Pro
Tyr Ser Leu Thr Ala Gln Gln Ser Ser Leu 50 55 60 atc aga gag gac
aag agc aac gcc aag ctg tgg aat gag gtc ctg gcg 240 Ile Arg Glu Asp
Lys Ser Asn Ala Lys Leu Trp Asn Glu Val Leu Ala 65 70 75 80 tca ctc
aag gac cgg ccg gcg agc ggc agc ccg ttc cag ttg ttc ctg 288 Ser Leu
Lys Asp Arg Pro Ala Ser Gly Ser Pro Phe Gln Leu Phe Leu 85 90 95
agt aaa gtg gag gag acg ttc cag tgt atc tgc tgt cag gag ctg gtg 336
Ser Lys Val Glu Glu Thr Phe Gln Cys Ile Cys Cys Gln Glu Leu Val 100
105 110 ttc cgg ccc atc acg acc gtg tgc cag cac aac gtg tgc aag gac
tgc 384 Phe Arg Pro Ile Thr Thr Val Cys Gln His Asn Val Cys Lys Asp
Cys 115 120 125 ctg gac aga tcc ttt cgg gca cag gtg ttc agc tgc cct
gcc tgc cgc 432 Leu Asp Arg Ser Phe Arg Ala Gln Val Phe Ser Cys Pro
Ala Cys Arg 130 135 140 tac gac
ctg ggc cgc agc tat gcc atg cag gtg aac cag cct ctg cag 480 Tyr Asp
Leu Gly Arg Ser Tyr Ala Met Gln Val Asn Gln Pro Leu Gln 145 150 155
160 acc gtc ctc aac cag ctc ttc ccc ggc tac ggc aat ggc cgg tga 525
Thr Val Leu Asn Gln Leu Phe Pro Gly Tyr Gly Asn Gly Arg 165 170 8
174 PRT Homo sapiens 8 Arg Glu Lys Glu Asn Ser Lys Arg Glu Glu Glu
Glu Gln Gln Glu Gly 1 5 10 15 Gly Phe Ala Ser Pro Arg Thr Gly Lys
Gly Lys Trp Lys Arg Lys Ser 20 25 30 Ala Gly Gly Gly Pro Ser Arg
Ala Gly Ser Pro Arg Arg Thr Ser Lys 35 40 45 Lys Thr Lys Val Glu
Pro Tyr Ser Leu Thr Ala Gln Gln Ser Ser Leu 50 55 60 Ile Arg Glu
Asp Lys Ser Asn Ala Lys Leu Trp Asn Glu Val Leu Ala 65 70 75 80 Ser
Leu Lys Asp Arg Pro Ala Ser Gly Ser Pro Phe Gln Leu Phe Leu 85 90
95 Ser Lys Val Glu Glu Thr Phe Gln Cys Ile Cys Cys Gln Glu Leu Val
100 105 110 Phe Arg Pro Ile Thr Thr Val Cys Gln His Asn Val Cys Lys
Asp Cys 115 120 125 Leu Asp Arg Ser Phe Arg Ala Gln Val Phe Ser Cys
Pro Ala Cys Arg 130 135 140 Tyr Asp Leu Gly Arg Ser Tyr Ala Met Gln
Val Asn Gln Pro Leu Gln 145 150 155 160 Thr Val Leu Asn Gln Leu Phe
Pro Gly Tyr Gly Asn Gly Arg 165 170 9 324 DNA Homo sapiens 9
atgtggatcc aggttcggac catggatggg aggcagaccc acacggtgga ctcgctgtcc
60 aggctgacca aggtggagga gctgaggcgg aagatccagg agctgttcca
cgtggagcca 120 ggcctgcaga ggctgttcta caggggcaaa cagatggagg
acggccatac cctcttcgac 180 tacgaggtcc gcctgaatga caccatccag
ctcctggtcc gccagagcct cgtgctcccc 240 cacagcacca aggagcggga
ctccgagctc tccgacaccg actccggctg ctgcctgggc 300 cagagtgagt
cagacaagtc ctcc 324 10 495 DNA Homo sapiens 10 gaggacatgt
gggatgagac ggaattgggg ctgtacaagg tcaatgagta cgtcgatgct 60
cgggacacga acatgggggc gtggtttgag gcgcaggtgg tcagggtgac gcggaaggcc
120 ccctcccggg acgagccctg cagctccacg tccaggccgg cgctggagga
ggacgtcatt 180 taccacgtga aatacgacga ctacccggag aacggcgtgg
tccagatgaa ctccagggac 240 gtccgagcgc gcgcccgcac catcatcaag
tggcaggacc tggaggtggg ccaggtggtc 300 atgctcaact acaaccccga
caaccccaag gagcggggct tctggtacga cgcggagatc 360 tccaggaagc
gcgagaccag gacggcgcgg gaactctacg ccaacgtggt gctgggggat 420
gattctctga acgactgtcg gatcatcttc gtggacgaag tcttcaagat tgagcggccg
480 ggtgaaggga gcccc 495 11 915 DNA Homo sapiens 11 gtctgcgcct
gccacctgtg cgggggccgg caggaccccg acaagcagct catgtgcgat 60
gagtgcgaca tggccttcca catctactgc ctggacccgc ccctcagcag tgttcccagc
120 gaggacgagt ggtactgccc tgagtgccgg aatgatgcca gcgaggtggt
actggcggga 180 gagcggctga gagagagcaa gaagaatgcg aagatggcct
cggccacatc gtcctcacag 240 cgggactggg gcaagggcat ggcctgtgtg
ggccgcacca aggaatgtac catcgtcccg 300 tccaaccact acggacccat
cccggggatc cccgtgggca ccatgtggcg gttccgagtc 360 caggtcagcg
agtcgggtgt ccatcggccc cacgtggctg gcatccatgg ccggagcaac 420
gacggatcgt actccctagt cctggcgggg ggctatgagg atgatgtgga ccatgggaat
480 tttttcacat acacgggtag tggtggtcga gatctttccg gcaacaagag
gaccgcggaa 540 cagtcttgtg atcagaaact caccaacacc aacagggcgc
tggctctcaa ctgctttgct 600 cccatcaatg accaagaagg ggccgaggcc
aaggactggc ggtcggggaa gccggtcagg 660 gtggtgcgca atgtcaaggg
tggcaagaat agcaagtacg cccccgctga gggcaaccgc 720 tacgatggca
tctacaaggt tgtgaaatac tggcccgaga aggggaagtc cgggtttctc 780
gtgtggcgct accttctgcg gagggacgat gatgagcctg gcccttggac gaaggagggg
840 aaggaccgga tcaagaagct ggggctgacc atgcagtatc cagaaggcta
cctggaagcc 900 ctggccaacc gagag 915 12 1369 DNA Homo sapiens 12
ggcagcgttt gccgagcggg cgctccgggt cgcacgcaag tccgcgcggg gtccgggcca
60 cgcacgcggt ttcatcgcca tccccagccg ggccaggcgc gcaggcagac
aagctgttcg 120 cggcgaccgg agaggtgagc gggcgggccg ggtcggggtg
ccagcccggg ccgggcgcac 180 ggggctcggg aactttgcaa aactttcccg
cgcggccagc ccgggcgcac gcatgtcccg 240 cactctgtcc cgggatccag
ggcctcccct tccacctaac cctcgggaat cgttccccgg 300 cacacatccg
gctggagccg ggaccagcgc tgcgtccccg gagcccggcg gggggtcgag 360
cgcgccgggt gggggagggc ctggcgagcc gccggggagg atgtcaggct ccgcgcctgc
420 gcgcggggcg ccccgcgatt caattgtcgc gcccgagccc gatttcgcgc
gccctgagtt 480 ccccgggagc atctgggcca atggggagcg agcggggcgg
ggcggccggg tgctgcggag 540 ccaataagag gcggctcaag tgaagggggg
cgggacttga cgagcggggg ccccctctgt 600 agtcccggcg gcgggggtgg
gcgtgggctc gctggcgcga cccgcgcggg ccagtgggag 660 tgcgggaggg
acgccgaggg tccagggttt ggaggggcgc gagctgccgg gggttggagg 720
tcgaggtgag tcgcggggcg cgcgcgctcg cgggtggccg ggacggggcg cggttaccat
780 ggccaccgcg gggcgggccc ggtcgcgcac gcgcgcgggg ggggccggca
aggagggggg 840 gcgtgggcac cgaggggtcc cggggtccgc ggatctcggg
tggggttttt cccatttcag 900 tggcacttgg ttaagttccc ccgggacctt
ctgaagttcc ggcccgcgct ggactttctg 960 ggattccctc ttccgtaaat
aggaatccga ggaatgaatg aatcaatgaa tgaatgaata 1020 aacgaaccaa
ctcgggccac ttggcccggg cctcctttct cctctggtcg tggggaagga 1080
gggatgggtt ggaccttctg cttttctttc aattccctct tttcattctc cttcctcctc
1140 aatcttcaac acttggctag tcgttaatgc cttaagtgct taatttgttg
tgtctggtcc 1200 tggccagggt ctggctgtac aggaggactg gaagggcatc
ctgggagttt cctggtgtcc 1260 acaggccgga caaaagcaac cccgactcct
tagagcatgg catggctcag aggtgctggt 1320 aaaactgatg ggggtttatg
ctgtccctcc cctcagcgcc gacaccatg 1369 13 36 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 13
aattcgattg gttctgattg gttctgattg gttctt 36 14 36 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 14 ctagaagaac caatcagaac caatcagaac caatcg 36 15 38
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 15 aattcggggc ggggccgggg cgggcccggg
gcggggct 38 16 39 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide 16 ctagagcccc gccccggccc
cgccccggcc ccgccccgg 39 17 21 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 17 agtcagggat
tggctggtct g 21 18 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 18 cagaccagcc
aatccctgac t 21 19 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 19 aagctacgat
tggttcttct g 21 20 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 20 cagaagaacc
aatcgtagct t 21 21 38 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 21 ataaaggcaa
gctacgattg gttcttctgg acggagac 38 22 39 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 22
gtctccgtcc agaagaacca atcgtagctt gccttttat 39 23 39 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 23 gaattcgagg gtaaaggggc ggggttgagg cagatgcca 39 24
38 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 24 tggcatctgc ctcaaccccg ccccttaccc
tcgaattc 38
* * * * *