U.S. patent application number 10/536257 was filed with the patent office on 2006-03-02 for novel use of aim 3 acting as a tumor suppressor.
Invention is credited to Sunghoon Kim.
Application Number | 20060046250 10/536257 |
Document ID | / |
Family ID | 35196744 |
Filed Date | 2006-03-02 |
United States Patent
Application |
20060046250 |
Kind Code |
A1 |
Kim; Sunghoon |
March 2, 2006 |
Novel use of aim 3 acting as a tumor suppressor
Abstract
The present invention relates to novel uses of AIM3 acting as a
tumor suppressor, and more particularly to methods for using an
AIM3 protein or a nucleic acid encoding the protein to activate ATM
or ATR and to treat ATM- or ATR-mediated diseases. The AIM3 protein
according to the present invention interacts directly with ATM/ATR
so as to activate ATM/ATR and proteins regulated by ATM/ATR. Also,
the AIM3 protein upregulates tumor suppressor gene p53 and its
target genes so as to not only inhibit the proliferation of cells
but also to induce apoptosis.
Inventors: |
Kim; Sunghoon; (Seoul,
KR) |
Correspondence
Address: |
BUCHANAN INGERSOLL PC;(INCLUDING BURNS, DOANE, SWECKER & MATHIS)
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Family ID: |
35196744 |
Appl. No.: |
10/536257 |
Filed: |
September 1, 2004 |
PCT Filed: |
September 1, 2004 |
PCT NO: |
PCT/KR04/02202 |
371 Date: |
May 25, 2005 |
Current U.S.
Class: |
435/6.18 ;
435/199; 435/320.1; 435/325; 435/69.1; 514/44R; 536/23.2 |
Current CPC
Class: |
A61P 13/08 20180101;
A61P 13/10 20180101; A61P 35/00 20180101; A61P 43/00 20180101; A61K
48/00 20130101; A61P 17/06 20180101; G01N 2333/4703 20130101; G01N
33/6893 20130101; C07K 14/4702 20130101; G01N 2500/04 20130101;
A61K 38/53 20130101; C07K 14/47 20130101; A61K 38/1709 20130101;
A01K 2217/054 20130101; A61P 35/02 20180101; G01N 2800/205
20130101; C12N 9/93 20130101; G01N 33/574 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/199; 435/320.1; 435/325; 514/044; 536/023.2 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12Q 1/68 20060101 C12Q001/68; C07H 21/04 20060101
C07H021/04; C12N 9/22 20060101 C12N009/22 |
Claims
1. A method for activating one selected from ATM, ATR and proteins
regulated by ATM or ATR, in the cell, tissue or individual,
comprising administering to the cell, tissue or individual an
effective amount of one selected from the group consisting of the
following: (a) an isolated polypeptide of AIM3 (ARS-interacting
multifunctional protein 3) having an amino acid sequence shown in
SEQ ID NO: 1; (b) an isolated polypeptide having an amino acid
sequence homology of at least 70% with the polypeptide (a); and (c)
an isolated nucleic acid encoding the polypeptide (a) or (b).
2. The method of claim 1, wherein the activation of ATM or ATR is
mediated by the binding of AIM3.
3. The method of claim 1, wherein the proteins regulated by ATM or
ATR are selected from the group consisting of H2AX, p53, chk2,
chk1, BRC AI, c-Abl, PHAS-1, RPA, RAD9, MDM2, MRE11, Rad17, WRN,
PTS, CtIP, eIF-4E binding protein 1, LKB1, FANCD2, SMCl, Rad17,
Nibrin, NBS, p95, Pin2/TRF1, DNA 5B, BRC A2 and
phosphatidylinositol 3-kinase.
4. The method of claim 1, wherein the activation of ATM, ATR and
proteins regulated by ATM or ATR is related with DNA repair, cell
cycle regulation and/or apoptosis.
5. A method for inducing the expression of p53 or its target genes
in the cell, tissue or individual, comprising administering to the
cell, tissue or individual an effective amount of one selected from
the group consisting of the following: (a) an isolated polypeptide
of AIM3 (ARS-interacting multifunctional protein 3) having an amino
acid sequence shown in SEQ ID NO: 1; (b) an isolated polypeptide
having an amino acid sequence homology of at least 70% with the
polypeptide (a); and (c) an isolated nucleic acid encoding the
polypeptide (a) or (b).
6. The method of claim 5, wherein the target genes of p53 are
selected from the group consisting of p21, PUMA, GADD45, 14-3-3
sigma, WIPI, mdm-2, EGFR, PCNA, Cyclin D1, Cyclin G, TGFA, BAX,
BAK, FAS1, Fas/APO1, FASL, IGF-BP3, PAG608, DR5/KILLER, GML,
p53AIP1, p53R2, P2XM, TSP-1, BAL1, CSR, PIG3, Apaf-1, p53RDL1,
Staf50, CD200 and Snk/PIk2.
7. A method for inhibiting the proliferation of tumor cells,
comprising administering to the cell, tissue or individual an
effective amount of one selected from the group consisting of the
following: (a) an isolated polypeptide of AIM3 (ARS-interacting
multifunctional protein 3) having an amino acid sequence shown in
SEQ ID NO: 1; (b) an isolated polypeptide having an amino acid
sequence homology of at least 70% with the polypeptide (a); and (c)
an isolated nucleic acid encoding the polypeptide (a) or (b).
8. A method for stimulating apoptosis in the cell, tissue or body,
comprising administering to the cell, tissue or individual an
effective amount of one selected from the group consisting of the
following: (a) an isolated polypeptide of AIM3 (ARS-interacting
multifunctional protein 3) having an amino acid sequence shown in
SEQ ID NO: 1; (b) an isolated polypeptide having an amino acid
sequence homology of at least 70% with the polypeptide (a); and (c)
an isolated nucleic acid encoding the polypeptide (a) or (b).
9. A method for treating or preventing ATM- or ATR-mediated
diseases, comprising administering to a subject in need thereof an
effective amount of one selected from the group consisting of the
following: (a) an isolated polypeptide of AIM3 (ARS-interacting
multifunctional protein 3) having an amino acid sequence shown in
SEQ ID NO: 1; (b) an isolated polypeptide having an amino acid
sequence homology of at least 70% with the polypeptide (a); and (c)
an isolated nucleic acid encoding the polypeptide (a) or (b).
10. The method of claim 9, wherein the diseases are cancers or
psoriasis.
11. The method of claim 10, wherein the cancers include breast
cancer, rectal cancer, lung cancer, small-cell lung cancer, stomach
cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer,
skin cancer, head or neck cancer, skin or intraocular melanoma,
uterine carcinoma, ovarian cancer, colorectal cancer, cancer neat
the anus, colon cancer, oviduct carcinoma, endometrial carcinoma,
cervical cancer, vaginal cancer, vulva carcinoma, Hodgkin's
disease, esophagus cancer, small intestinal tumor, endocrine gland
cancer, thyroid cancer, parathyroid cancer, adrenal cancer,
soft-tissue sarcoma, uterine cancer, penis cancer, prostate cancer,
chronic or acute leukemia, lymphocytic lymphoma, bladder cancer,
kidney or urethra cancer, kidney cell carcinoma, kidney pelvis
carcinoma, CNS tumor, primary CNS lymphoma, spinal tumor, brain
stem glioma, and pituitary adenoma, and combinations of one or more
thereof.
12. A method for screening a substance having the effect of
treating and/or preventing ATM- or ATR-mediated diseases,
comprising the steps of: (a) culturing AIM3 (ARS-interacting
multifunctional protein 3) or a recombinant cell expressing the
protein, together with a candidate substance; and (b) determining
the effect of the candidate substance on an increase in the
activity of AIM3 or intracellular level thereof.
13. The method of claim 12, wherein the ATM- or ATR-mediated
diseases are cancers or psoriasis.
14. A method for identifying a subject having the risk of ATM- or
ATR-mediated diseases, comprising the steps of: (a) measuring the
expression level of AIM3 (ARS-interacting multifunctional protein
3) in tissue sampled from a subject; and (b) comparing the AIM3
level in the tissue with a normal AIM3 level.
15. The method of claim 14, wherein the ATM- or ATR-mediated
diseases are cancers or psoriasis.
16. A kit for the diagnosis of ATM- or ATR-mediated diseases,
comprising one selected from a AIM3 protein-encoding nucleic acid,
its fragment, a peptide encoded by the nucleic acid or its
fragment, and an antibody to the peptide.
17. The method of claim 16, wherein the ATM- or ATR-mediated
diseases are cancers or psoriasis.
18. A pharmaceutical composition for activating one selected from
ATM, ATR and proteins regulated by ATM or ATR, in the cell, tissue
or individual, comprising, as an active ingredient, one selected
from the group consisting of the following: (a) an isolated
polypeptide of AIM3 having an amino acid sequence shown in SEQ ID
NO: 1; (b) an isolated polypeptide having an amino acid sequence
homology of at least 70% with the polypeptide (a); and (c) an
isolated nucleic acid encoding the polypeptide (a) or (b).
19. A pharmaceutical composition for inducing the expression of p53
or its target genes in the cell, tissue or individual, comprising,
as an active ingredient, one selected from the group consisting of
the following: (a) an isolated polypeptide of AIM3 having an amino
acid sequence shown in SEQ ID NO: 1; (b) an isolated polypeptide
having an amino acid sequence homology of at least 70% with the
polypeptide (a); and (c) an isolated nucleic acid encoding the
polypeptide (a) or (b).
20. A pharmaceutical composition for inhibiting the proliferation
of tumor cells, comprising, as an active ingredient, one selected
from the group consisting of the following: (a) an isolated
polypeptide of AIM3 having an amino acid sequence shown in SEQ ID
NO: 1; (b) an isolated polypeptide having an amino acid sequence
homology of at least 70% with the polypeptide (a); and (c) an
isolated nucleic acid encoding the polypeptide (a) or (b).
21. A pharmaceutical composition for stimulating apoptosis in the
cell, tissue or individual, comprising, as an active ingredient,
one selected from the group consisting of the following: (a) an
isolated polypeptide of AIM3 having an amino acid sequence shown in
SEQ ID NO: 1; (b) an isolated polypeptide having an amino acid
sequence homology of at least 70% with the polypeptide (a); and (c)
an isolated nucleic acid encoding the polypeptide (a) or (b).
22. A pharmaceutical composition for treating or preventing ATM- or
ATR-mediated diseases, comprising, as an active ingredient, one
selected from the group consisting of the following: (a) an
isolated polypeptide of AIM3 having an amino acid sequence shown in
SEQ ID NO: 1; (b) an isolated polypeptide having an amino acid
sequence homology of at least 70% with the polypeptide (a); and (c)
an isolated nucleic acid encoding the polypeptide (a) or (b).
23. An isolated polypeptide for use as an active therapeutic
substance, being selected from the group consisting of the
following: (a) an isolated polypeptide of AIM3 having an amino acid
sequence shown in SEQ ID NO: 1; and (b) an isolated polypeptide
having an amino acid sequence homology of at least 70% with said
polypeptide.
24. An isolated nucleic acid for use as an active therapeutic
substance, encoding an isolated polypeptide of AIM3 having an amino
acid sequence shown in SEQ ID NO: 1 or an isolated polypeptide
having a sequence homology of at least 70% with said polypeptide.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel tumor suppressor,
and particularly to a novel tumor suppressor that activates ATM or
ATR.
BACKGROUND ART
[0002] Cells have a variety of fail-safe mechanisms, one of which
is to arrest the cell division of damaged chromosomal DNA and to
repair the damage, thus preventing mutations from settling. When
chromosomal DNA damaged by UV and the like is continued to undergo
cell division in a condition where the damage is not repaired, the
damaged chromosomal DNA will be replicated so as to accumulate
mutations. This leads to an increase in the incidence of cancer
cells. Accordingly, when DNA is damaged, cells operate a process of
repairing the damage and an intracellular feedback mechanism of
arresting the cell division until the repair of DNA damage is over,
followed by inhibiting the development of cancers. Such a feedback
mechanism is mediated by checkpoints in each cycle of cell
division. The overall function of these checkpoints is to detect
damaged or abnormally structured DNA and to coordinate cell-cycle
progression with DNA repair (Robert T. Genes & development,
15:2177-2196, 2001). Typically, cell-cycle checkpoint activation
slows or arrests cell-cycle progression, thereby allowing time for
appropriate repair mechanisms to correct genetic lesions before
they are passed on to the next generation of daughter cells. In
certain cells, such as thymocytes, checkpoint proteins link DNA
strand breaks to apoptotic cell death via the induction of p53
(Robert T. Genes & development, 15:2177-2196, 2001).
[0003] Cell-cycle checkpoints which are initiated by DNA damages
are mainly regulated by ATM (ataxia-telangiectasia-mutated) and ATR
(ATM- and Rad3-ralated) proteins (Shiloh, Y. Curr. Opin. Gent.
Dev., 11:71-77, 2001; Abraham, R. T. Genes Dev., 15:2177-2196,
2001). Such proteins play a key role in the early signal
transduction via the cell-cycle checkpoints. ATM- and ATR-deficient
cells showed defects in arresting the cell cycle in response to
radiation. Particularly, the ATM-deficient cells showed serious
defects in G1, S and G2 checkpoints (Robert T. Genes &
development, 15:2177-2196, 2001), and serious damages called
"double strand breaks" occurred in the ATR-deficient cells.
Furthermore, it was known that the incidence of tumor is greatly
increased by the mutation of ATM/ATR.
[0004] ATM and ATR are highly homologous to each other and use the
same substrate. However, they are different in that their
activities are increased by different genotoxic stresses. ATM
responds to agents, such as IR (ionizing radiation) that breaks
double strands DNA, whereas ATR responds to agents (including IR)
that cause bulky adducts on DNA or single strand DNA. Furthermore,
ATM and ATR are activated by different methods. ATM activation
requires autophosphorylation that results in the disruption of an
ATM dimer (Bakkenist, C. J. et al., Nature, 421:499-506, 2003). How
autophosphorylation of ATM triggered is still unknown. ATR may also
be autophosphorylated, but it is not evident that ATR forms either
an inactive dimer or an active monomer in cells. Also, it is not
yet clear that other subunits or cofactors are required for the
activation of ATM/ATR. In addition, the intracellular biochemical
mechanism of a signal transduction system where the DNA damage
causes the activation and operation of ATM/ATR was not completely
established.
[0005] Target proteins known to be phosphorylated directly by
ATM/ATR include p53, chk1, chk2, c-Abl, RPA and the like, of which
p53 is phosphorylated on serine 15 by ATM/ATR. It was reported that
the over-expression of p53 arrests G2 and suppresses the synthesis
of two proteins, CDK1 (cyclin-dependent kinase 1) and cyclin B1,
which are required for the entry of cells from G2 to M. Thus, p53
does not only the function of inhibiting the abnormal division and
proliferation of cells, but also the function of arresting the cell
cycle so as to repair the damaged DNA when DNA was damaged.
Recently, the mutation and loss of p53 genes are recognized as one
of the most frequent genetic mutations, which is found not only in
any certain cancer but in almost all types of cancer in human.
Moreover, p53 activates the transcription of p21, another tumor
suppressor gene, thereby inhibiting the G1/S transition and causing
the p53-dependent apoptosis. p21 which is expressed by p53 was
known to be a kind of a CKI (cyclin-dependent kinase inhibitor)
which functions to inhibit the division and proliferation of cells.
Accordingly, efforts for developing new anticancer agents using
cell-cycle regulation factors or substances of activating the
factors are now continued.
[0006] Meanwhile, aminoacyl-tRNA synthetases (ARSs) which are
important enzymes catalyzing the first step in protein synthesis
are multifunctional proteins involved in various biological
functions (Ko et al., Proteomics, 2:1304-1310, 2002). Among them,
various mammalian tRNA synthetases, such as MRS (methionyl-tRNA
synthetase), QRS (glutaminyl-tRNA synthetase), RRS (arginyl-tRNA
synthetase), KRS (Lysyl-tRNA synthetase), DRS(aspartyl-tRNA
synthetase) and so on, bind to three non-enzyme cofactors,
designated as p43, p38 and p18, to form a macromolecular protein
complex (Han et al., Biochem. Biophys. Res. Commun., 303:985-993,
2003). Since ARSs are enzymes necessary for protein synthesis, this
complex deems to be formed in order to facilitate protein
synthesis.
[0007] Among the non-enzyme cofactors binding to ARSs, p43 is known
to play an important role as a cytokine in immune response and
angiogenesis (Ko et al., J. Biol. Chem., 276:23028-32303, 2001b;
Park et al., J. Biol. Chem., 277:45234-45248, 2002). Furthermore,
p38 was found to downregulate c-myc, a protoocogene, and to be
involved in lung differentiation (Kim et al., Nat. Genet.,
34:330-336, 2003). The last cofactor, p18, shows sequence homology
to elongation factor subunits (EF-1) (Quevillon and Mirande, FEBS
Lett., 395:63-67, 1996). Given this, p18 is presumed to be involved
in protein synthesis. However, the biological functions of p18 are
not yet clearly understood, and particularly, there is no study on
the relation between p18 and cancer.
DISCLOSURE OF THE INVENTION
[0008] Therefore, it is an object of the present invention to
provide novel uses of a p18 (ARS-interacting multifunctional
protein 3) protein.
[0009] The present inventors renamed p18 which had been known as a
cofactor of an aminoacyl-tRNA synthetase (ARS) complex to "AIM3
(ARS-interacting multifunctional protein 3)". Accordingly, p18 will
hereinafter be referred to as "AIM3".
[0010] To achieve the above object, in one aspect, the present
invention provides a method for activating ATM, ATR and proteins
regulated by ATM or ATR, in the cell, tissue and individual,
comprising administering to the cell, tissue or individual an
effective amount of one selected from the group consisting of the
following:
[0011] (a) an isolated polypeptide of AIM3 (ARS-interacting
multifunctional protein 3);
[0012] (b) an isolated polypeptide having at least 70% homology
with the polypeptide (a); and
[0013] (c) an isolated nucleic acid encoding the polypeptide (a) or
(b).
[0014] In another aspect, the present invention provides a method
for inducing the expression of p53 or its target genes in the cell,
tissue or individual, comprising administering to the cell, tissue
or individual an effective amount of one selected from the group
consisting of following:
[0015] (a) an isolated polypeptide of AIM3 protein;
[0016] (b) an isolated polypeptide having at least 70% homology
with the polypeptide (a); and
[0017] (c) an isolated nucleic acid encoding the polypeptide (a) or
(b).
[0018] In still another aspect, the present invention provides a
method for inhibiting the proliferation of tumor cells, comprising
administering to the cell, tissue or individual an effective amount
of one selected from the group consisting of the following:
[0019] (a) an isolated polypeptide of AIM3 protein;
[0020] (b) an isolated polypeptide having at least 70% homology
with the polypeptide (a); and
[0021] (c) an isolated nucleic acid encoding the polypeptide (a) or
(b).
[0022] In still another aspect, the present invention provides a
method for stimulating apoptosis in the cell, tissue or individual,
comprising administering to the cell, tissue or individual an
effective amount of one selected from the group consisting of the
following:
[0023] (a) an isolated polypeptide of AIM3 protein;
[0024] (b) an isolated polypeptide having at least 70% homology
with the polypeptide (a); and
[0025] (c) an isolated nucleic acid encoding the polypeptide (a) or
(b).
[0026] In still another aspect, the present invention provides a
method for treating or preventing ATM- or ATR-mediated diseases,
comprising administering to a subject in need thereof an effective
amount of one selected from the group consisting of the
following:
[0027] (a) an isolated polypeptide of AIM3 protein;
[0028] (b) an isolated polypeptide having at least 70% homology
with the polypeptide (a); and
[0029] (c) an isolated nucleic acid encoding the polypeptide (a) or
(b).
[0030] In still another aspect, the present invention provides a
method for screening a substance having the effect of treating or
preventing ATM- or ATR-mediated diseases, the method comprising the
steps of:
[0031] (a) culturing AIM3 (ARS-interacting multifunctional protein
3) or a recombinant cell expressing the protein, together with a
candidate substance; and
[0032] (b) determining the effect of the candidate substance on an
increase in the activity of AIM 3 or the intracellular level
thereof.
[0033] In yet another aspect, the present invention provides a
method for identifying a subject having the risk of ATM- or
ATR-mediated diseases, comprising the steps of:
[0034] (a) measuring the expression level of ATM3 protein in tissue
sampled from a subject; and
[0035] (b) comparing the level of the AIM3 protein in the tissue
with a normal AIM3 protein level.
[0036] In still another aspect, the present invention provides a
kit for the diagnosis of ATM- or ATR-mediated diseases, comprising
one selected from the group consisting of an AIM3 protein-encoding
nucleic acid, a fragment thereof, a peptide encoded by the nucleic
acid or its fragment, and an antibody to the peptide.
[0037] In another further aspect, the present invention provides
pharmaceutical compositions comprising, as an active ingredient,
one selected from the group consisting of the following:
[0038] (a) an isolated polypeptide of AIM3 protein;
[0039] (b) an isolated polypeptide having at least 70% homology
with the polypeptide (a); and
[0040] (c) an isolated nucleic acid encoding the polypeptide (a) or
(b).
[0041] Hereinafter, the present invention will be described in
detail.
[0042] In the present invention, novel activities of AIM3 (p18)
known as a cofactor of an aminoacyl-tRNA synthetase (ARS) complex
were identified. The physiological activities (functions) of AIM3
identified in the present invention are as follows:
[0043] First, in the DNA synthesis step and upon DNA damage, AIM3
is moved into nuclei and induced at a high level.
[0044] Second, AIM3 shows an anti-proliferation activity against
cells.
[0045] Third, AIM3 induces apoptosis.
[0046] Fourth, AIM3 induces the expression of tumor suppressor gene
p53 and its target genes.
[0047] Fifth, AIM3 interacts directly with ATM/ATR so as to
activate ATM, ATR and proteins which are regulated by ATM or
ATR.
[0048] Sixth, a reduction in the expression level of AIM3 induces
tumorigenesis, and it is expressed at a low level in cancer cell
lines and tissues isolated from cancer patients.
[0049] Accordingly, the present invention provides a method for
activating one selected from the group consisting of ATM, ATR and
proteins regulated by ATM or ATR using an AIM3 protein or a nucleic
acid encoding the AIM3 protein.
[0050] As used herein, the term "activating" means the
phosphorylation of proteins or the structural or chemical mutation
of proteins. The activation of ATM/ATR is mediated by the biding of
AIM3, which causes a variety of intracellular responses involved in
ATM/ATR. Such intracellular responses include, but are not limited
to, DNA repair, cell cycle regulation and apoptosis induction.
Thus, the activation of ATM/ATR by AIM3 accompanies activation of
downstream proteins which are involved in DNA repair signal
transduction pathways induced by DNA replication or damage, a
checkpoint signal transduction pathway in each cell cycle, and/or
an apoptosis-inducing signal transduction pathway caused by DNA
damage. The ATM/ATR-regulated proteins include proteins which are
directly phosphorylated by ATM/ATR, and proteins which are
sequentially phosphorylated in signal transduction pathways by the
phosphorylation of said proteins. Preferred examples include, but
are not limited to, H2AX (Burma S. et al., J. Biol. Chem.,
9;276(45):42462-42467, 2001), p53 (Saito S, et al., J. Biol. Chem.,
12;277(15):12491-12494, 2002), chk2 (Matsuoka S, et al., Proc.
Natl. Acad. Sci. U.S.A., 12;97(19):10389-10394, 2000), chk1 (Kim S
T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999), BRCA1 (Xu
B, et al., Cancer Res., 15;62(16):4588-4591, 2002; Cortez D, et
al., Science, 5;286(5442):1162-1166, 1999), c-Abl (Baskaran R, et
al., Nature, 29;387(6632):516-519, 1997), PHAS-1 (Chan D W et al.,
J. Biol. Chem., 17;275(11):7803-7810, 2000), RPA (Chan D W et al.,
J. Biol. Chem., 17;275(11):7803-7810, 2000), RAD9 (Chen M J et al.,
J. Biol. Chem., 1 1;276(19):16580-16586, 2001), MDM2 (Maya R, et
al., Genes Dev., 1;15(9):1067-1077, 2001), MRE11 (Kim S T et al.,
J. Biol. Chem., 31;274(53):37538-37543, 1999), Rad17 (Kim S T et
al., J. Biol. Chem., 31;274(53):37538-37543, 1999), WRN (Kim S T et
al., J. Biol. Chem., 31;274(53):37538-37543, 1999), PTS (Kim S T et
al., J. Biol. Chem., 31;274(53):37538-37543, 1999), CtIP (Li S, et
al., Nature, 13;406(6792):210-215, 2000), eIF-4E binding protein 1
(Yang D Q, et al., Nat. Cell. Biol., 2(12):893-898, 2000), LKB1
(Sapkota G P, et al., Biochem J., 1;368(Pt 2):507-516, 2002),
FANCD2 (Taniguchi T, et al., Cell, 17;109(4):459-472, 2002), SMC1
(Yazdi P T, et al., Genes Dev., 1;16(5):571-582, 2002), Rad17 (Kim
S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999), Nibrin
(Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999), NBS
(Wu K. et al., Nature, 25;405(6785):477-482, 2000), p95 (Kim S T et
al., J. Biol. Chem., 31;274(53):37538-37543, 1999), Pin2/TRF1
(Kishi S. et al., J. Biol. Chem., 3;276(31):29282-29291, 2001), DNA
5B (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543, 1999),
BRCA2 (Kim S T et al., J. Biol. Chem., 31;274(53):37538-37543,
1999) and phosphatidylinositol 3-kinase (Kim S T et al., J. Biol.
Chem., 31;274(53):37538-37543, 1999). More preferably, the proteins
may be H2AX, p53 or chk2.
[0051] Furthermore, the present invention provides a method for
inducing the expression of p53 or its target gene using the AIM3
protein or a nucleic acid encoding the AIM3 protein. As used
herein, the term "p53-target gene" refers to a gene located in
downstream of p53, whose expression is induced by p53. The
p53-target gene may be a gene involved in at least one mechanism
selected from the group consisting of p53 control, cell cycle
regulation, DNA repair, apoptosis, angiogenesis, cellular stress
response and determination of cell fate. Preferred examples of this
target gene include, but are not limited to, p21 (Fujioka S, et
al., J. Biol. Chem., Apr. 21, 2004; Nayak B K, et al., Oncogene,
17;21(47):7226-7229, 2002), PUMA (Gu J, et al., Oncogene,
12;23(6):1300-1307, 2004; Yu J, et al., Cell, 7(3):673-682, 2001),
GADD45 (Nayak B K, et al., Oncogene, 17;21(47):7226-7229, 2002;
el-Deiry W S., Semin Cancer Biol., 8(5):345-57), 14-3-3 sigma
(el-Deiry W S., Semin Cancer Biol., 8(5):345-57), WIP1 (Choi J, et
al., Genomics., 15;64(3):298-306, 2000), mdm-2 (Freedman and
Levine, Cancer Research, 59:1-7, 1999), EGFR (Tokino and Nakamura,
Crit. Rev. Onc. Hem., 33:1-6, 2000), PCNA (Tokino and Nakamura,
Crit. Rev. Onc. Hem., 33:1-6, 2000), Cyclin D1 (Tokino and
Nakamura, Crit. Rev. Onc. Hem., 33:1-6, 2000), Cyclin G (Tokino and
Nakamura, Crit. Rev. Onc. Hem., 33:1-6, 2000), TGF.alpha. (Inoue Y,
et al., Hepatology, 36(2):366-344, 2002), BAX (Gu J, et al.,
Oncogene, 12;23(6):1300-1307, 2004; Nayak B K, et al., Oncogene,
17;21(47):7226-7229, 2002), BAK (Gu J, et al., Oncogene,
12;23(6):1300-1307, 2004), FAS1 (Gu J, et al., Oncogene,
12;23(6):1300-1307, 2004), Fas/APO1 (el-Deiry W S., Semin Cancer
Biol., 8(5):345-57), FASL (Mendoza-Rodriguez C A, et al., Rev.
Invest. Clin., 53(3):266-273, 2001), IGF-BP3 (Mendoza-Rodriguez C
A, et al., Rev. Invest. Clin., 53(3):266-273, 2001), PAG608
(Higashi Y, et al., J. Biol. Chem., 1;277(44):42224-42262, 2002),
DR5/KILLER (Takimoto R, et al., Oncogene, 30;19(14):1735-1743,
2000), GML (Higashiyama M, et al., Eur. J. Cancer., 36(4):489-495,
2000; Nakamura Y., Cancer Sci., 95(1):7-11, 2004), p53AIP1
(Nakamura Y., Cancer Sci., 95(1):7-11, 2004), STAG1 (Nakamura Y.,
Cancer Sci., 95(1):7-11, 2004), p53R2 (Nakamura Y., Cancer Sci.,
95(1):7-11, 2004), p53RFP (Nakamura Y., Cancer Sci., 95(1):7-11,
2004), P2XM (Nawa G, et al., Br. J. Cancer., 80(8):1185-1189,
1999), TSP-1 (Harada H, et al., Cancer Lett., 28;191(1):109-119,
2003), BAL1 (Nakamura Y., Cancer Sci., 95(1):7-11, 2004), CSR
(Nakamura Y., Cancer Sci., 95(1):7-11, 2004), PIG3 (Giampieri S, et
al., Oncogene, Apr. 12, 2004; Contente A, et al., Nat. Genet.,
30(3):315-320, 2002), Apaf-1 (Giampieri S, et al., Oncogene, Apr.
12, 2004), p53RDL1 (Nakamura Y., Cancer Sci., 95(1):7-11, 2004),
Staf50 (Obad S, et al., Oncogene, 20;23(3):4050-4059, 2004), CD200
(Rosenblum M D, et al., Blood, 1;103(7):2691-2698, 2004) and
Snk/PIk2 (Bums T F, et al., Mol. Cell. Biol., 23(16):5556-5571,
2003). More preferably, the target gene may be p21 or PUMA.
[0052] AIM3 of the present invention inhibits the proliferation of
tumor cells through signal transduction pathways mediated by
ATM/ATR and stimulates apoptosis caused by DNA damage. Accordingly,
the present invention provides methods to inhibit the proliferation
of tumor cells and to stimulate apoptosis, using the AIM3 protein
and a nucleic acid encoding the AIM3 protein.
[0053] All the methods described above comprise administering an
effective amount of the AIM3 protein or the nucleic acid encoding
the protein to cells or tissues. As used herein, the term
"effective amount" refers to the amount of AIM3, which shows an
effect selected from the group consisting of the following: the
activation of ATM/ATR in cells or tissues; the increase of
phosphorylation of ATM/ATR-regulated proteins; the induction of
expression of p53 or its target gene; the inhibition of
proliferation of tumor cells; and the promotion of apoptosis.
[0054] The AIM3 proteins used in the present invention include
natural or recombinant AIM3 proteins, or proteins having the
substantially equivalent physiological activity of the natural or
recombinant AIM3 proteins. The amino acid sequence of the AIM3
protein is known in the art and preferably derived from mammals,
including human beings. The AIM3 protein of the present invention
preferably has an amino acid sequence shown in SEQ ID NO: 1.
Proteins having the substantially equivalent physiological activity
of AIM3 include natural/recombinant AIM3 proteins, their functional
equivalents and their functional derivatives. As used herein, the
term "the substantially equivalent physiological activity" means
the activity of: activating ATM/ATR or ATM/ATR-regulated proteins;
inducing the expression of p53 or its target gene; inhibiting the
proliferation of tumor cells; and/or stimulating apoptosis. The
term "functional equivalents" refers to amino acid sequence
variants with a substitution of some or all of the amino acids of a
natural AIM3 protein or a deletion or addition of some of the amino
acids, which have a physiological activity substantially equivalent
to the natural AIM3 protein. Furthermore, the term "functional
derivatives" refers to those having a physiological activity
substantially equivalent to natural AIM3 protein, as proteins
modified to increase or reduce the physicochemical properties of
the AIM 3 protein. The proteins having a physiological activity
substantially equivalent to the AIM3 protein have a homology of at
least 70%, preferably at least 80%, and more preferably at least
90%, with the polypeptide shown in SEQ ID NO: 1. The AIM3 protein
used in the present invention can be prepared by any genetic
engineering method known in the art.
[0055] The inventive pharmaceutical composition containing the AIM3
protein as an active ingredient can be administered to human beings
and animals by oral route or by parenteral route, such as an
intravenous, subcutaneous, intranasal or intraperitoneal route.
Oral administrations include sublingual application. Parenteral
administrations include injection techniques, such as subcutaneous
injection, intramuscular injection and intravenous injection, as
well as drip infusion. In addition, the pharmaceutical composition
can be formulated into various forms with a pharmaceutically
acceptable carrier by a conventional method. As used herein, the
term "pharmaceutically acceptable" carrier means a substance which
is physiologically acceptable and, when administered to human
beings, generally does not cause allergic reactions, such as
gastrointestinal disorder and dizziness, or similar reactions
thereto.
[0056] As the pharmaceutically acceptable carriers, in the case of
oral administration, there may be used binders, lubricants,
disintegrants, excipients, solubilizers, dispersing agents,
stabilizers, suspension agents, pigments and flavors, and in case
of injection agent, there can be used buffers, preservatives,
analgesics, solubilizers, isotonics and stabilizers, and in case of
formulations for local administration may include bases,
excipients, lubricants and preservatives. As described above, the
inventive pharmaceutical composition containing the AIM3 protein
may be formulated into various forms with the pharmaceutically
acceptable carriers. For example, for oral administration, the
inventive composition may be formulated into the form of tablets,
troches, capsules, elixirs, suspensions, syrups, wafers and so on,
and for injection agent, it may be formulated into unit dose
ampoules or multiple dose products.
[0057] A total effective amount of the AIM3 protein of the present
invention can be administered to patients in a single dose or can
be administered by a fractionated treatment protocol, in which
multiple doses are administered over a more prolonged, period of
time. Although the amount of the AIM3 protein or a nucleic acid
encoding the AIM3 protein in the inventive pharmaceutical
composition may vary depending on the severity of diseases, the
protein or the nucleic acid may be generally administered several
times a day at an effective dose of 1 .mu.g-10 mg. However, a
suitable dose of the AIM3 protein in the inventive pharmaceutical
composition may depend on many factors, such as the age, body
weight, health condition, sex, disease severity, diet and excretion
of patients, as well as the route of administration and the number
of treatments to be administered. In view of these factors, any
person skilled in the art may determine an effective dose for
treating or preventing ATM/ATR-mediated diseases. The inventive
pharmaceutical composition containing the AIM3 protein has no
special limitations on its formulation, administration route and/or
administration mode insofar as it shows the effects of the present
invention.
[0058] Meanwhile, nucleic acids encoding the AIM3 protein of the
present invention include DNA or RNA. Preferably, they refer to DNA
encoding AIM3 proteins derived from mammals, particularly human
beings. The human AIM3 gene is known in the art (GenBank accession
No. AB011079). Preferably, the nucleic acid of the present
invention is shown in SEQ ID NO: 2. The nucleic acids also include
nucleic acids encoding functional equivalents to the AIM3 protein.
The present invention can be included nucleic acids having a
sequence homology of at least 80%, preferably at least 90%, and
more preferably at least 95% with either a nucleic acid encoding
the AIM3 protein or a nucleic acid comprising the complementary
nucleotide sequence thereof.
[0059] The nucleic acid encoding the AIM3 protein may be used for
gene therapy by inserting it into an expression vector, such as a
plasmid or viral vector, and then introducing the expression vector
into a target cell by any method known in the art, such as
infection or transduction.
[0060] A gene transfer method using a plasmid expression vector is
a method of transferring a plasmid DNA directly to human cells,
which is an FDA-approved method applicable to human beings (Nabel,
E. G., et al., Science, 249:1285-1288, 1990). Unlike viral vectors,
the plasmid DNA has an advantage of being homogeneously purified.
Plasmid expression vectors which can be used in the present
invention include mammalian expression plasmids known in the
pertinent art. For example, they are not limited to, but typically
include pRK5 (European Patent No. 307,247), pSV16B (PCT Publication
No. 91/08291) and pVL1392 (PharMingen).
[0061] The plasmid expression vector containing the nucleic acid
according to the present invention may be introduced into target
cells by any method known in the art, including, but not limited
to, transient transfection, microinjection, transduction, cell
fusion, calcium phosphate precipitation, liposome-mediated
transfection, DEAE dextran-mediated transfection,
polybrene-mediated transfection, electroporation, gene gun methods,
and other known methods for introducing DNA into cells (Wu et al.,
J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem.,
263:14621-14624, 1988).
[0062] In addition, virus expression vectors containing the nucleic
acid according to the present invention include, but are not
limited to, retrovirus, adenovirus, herpes virus, avipox virus and
so on.
[0063] The retroviral vector is so constructed that non-viral
proteins can be produced within the infected cells by the viral
vector in which virus genes are all removed or modified. The main
advantages of the retroviral vector for gene therapy are that it
transfers a large amount of genes into replicative cells, precisely
integrates the transferred genes into cellular DNA, and does not
induce continuous infections after gene transfection (Miller, A.
D., Nature, 357:455-460, 1992). The retroviral vector approved by
FDA was prepared using PA317 amphotropic retrovirus packaging cells
(Miller, A. D. and Buttimore, C., Molec. Cell Biol., 6:2895-2902,
1986).
[0064] Non-retroviral vectors include adenovirus as described above
(Rosenfeld et al., Cell, 68:143-155, 1992; Jaffe et al., Nature
Genetics, 1:372-378, 1992; Lemarchand et al., Proc. Natl. Acad.
Sci. USA, 89:6482-6486, 1992). The main advantages of adenovirus
are that it transfers a large amount of DNA fragments (36 kb
genomes) and is capable of infecting non-replicative cells at a
very high titer.
[0065] Moreover, herpes virus may also be useful for human genetic
therapy (Wolfe, J. H., et al., Nature Genetics, 1:379-384, 1992).
In addition, any suitable virus vector known in the art may be
used.
[0066] A vector capable of expressing the AIM3 gene may be
administered by a known method. For example, the vector may be
administered locally, parenterally, orally, intranasally,
intravenously, intramuscularly or subcutaneously, or by other
suitable routes. Particularly, the vector may be injected directly
into a target cancer or tumor cell at an effective amount for
treating the tumor cell of a target tissue. Particularly for a
cancer or tumor present in a body cavity such as in the eye,
gastrointestinal tract, genitourinary tract, pulmonary and
bronchial system and so on, the inventive pharmaceutical
composition can be injected directly into the hollow organ affected
by the cancer or tumor using a needle, a catheter or other delivery
tubes. Any effective imaging device, such as X-ray, sonogram, or
fiberoptic visualization system, may be used to locate the target
tissue and guide the needle or catheter tube. In addition, the
inventive pharmaceutical composition comprising the nucleic acid
encoding the AIM3 protein may be administered into the blood
circulation system for treatment of a cancer or tumor which cannot
be directly reached or anatomically isolated.
[0067] The pharmaceutical composition comprising the nucleic acid
encoding the AIM3 protein as an active ingredient may additionally
comprise pharmaceutically acceptable carriers or excipients. These
carriers or excipients include dispersing agents, wetting agents,
suspending agents, diluents and fillers. The ratio of the
particular pharmaceutically acceptable carrier and the expression
vector contained in the inventive pharmaceutical composition can be
determined by the solubility and chemical properties of the
composition, and the particular administration mode. The
therapeutic or preventive effective amount of the inventive
pharmaceutical composition containing the AIM3 protein-encoding
nucleic acid may be suitably selected depending on the subject to
be administered, age, individual variation and disease
condition.
[0068] In another aspect, the present invention provides a method
for treating or preventing ATM/ATR-mediated diseases using the AIM3
protein or a nucleic acid encoding the AIM3 protein. Specifically,
the present invention provides a method for treating or preventing
ATM- or ATR-mediated diseases, which comprise administering to a
subject requiring treatment an effective amount of one selected
from the group consisting of the following: (a) an isolated
polypeptide of an AIM3 protein; (b) a polypeptide having at least
70% homology with the polypeptide (a); and (c) an isolated nucleic
acid encoding the polypeptide (a) or (b). As used herein, the term
"subject" means mammals, particularly animals including human
beings. The subject may be a patient requiring treatment.
Furthermore, the term "ATM- or ATR-mediated diseases" refers to
diseases induced by the inactivation or activation reduction of
ATM/ATR, i.e., diseases induced by abnormalities occurring in
signal transduction pathways mediated by ATM/ATR, due to the
inactivation or activation reduction of ATM/ATR. The signal
transduction pathways mediated by ATM/ATR include signal
transduction pathways mediated by ATM/ATR themselves or
ATM/ATR-regulated proteins. The signal transduction pathways may be
signal transduction pathways in DNA repair, cell cycle regulation,
apoptosis, p53 regulation, angiogenesis and/or intracellular stress
response. The ATM/ATR-mediated diseases may be caused by the
over-proliferation of cells, such as cancers or psoriasis. The
cancers include, but are not limited to, breast cancer, rectal
cancer, lung cancer, small-cell lung cancer, stomach cancer, liver
cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer,
head or neck cancer, skin or intraocular melanoma, uterine
carcinoma, ovarian cancer, colorectal cancer, cancer near the anus,
colon cancer, oviduct carcinoma, endometrial carcinoma, cervical
cancer, vaginal cancer, vulva carcinoma, Hodgkin's disease,
esophagus cancer, small intestinal tumor, endocrine gland cancer,
thyroid cancer, parathyroid cancer, adrenal cancer, soft-tissue
sarcoma, uterine cancer, penis cancer, prostate cancer, chronic or
acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or
urethra cancer, kidney cell carcinoma, kidney pelvis carcinoma, CNS
tumor, primary CNS lymphoma, spinal tumor, brain stem glioma, and
pituitary adenoma, and a combination of one or more thereof.
Particularly, the treating or preventing method according to the
present invention is effective in treating or preventing cancers
caused by p53 gene abnormalities. In this method, the dose
(effective amount) and administration mode of the AIM3 protein or
the nucleic acid encoding the AIM3 protein are the same as
described above.
[0069] The AIM3 protein of the present invention interacts directly
with ATM/ATR so as to activate ATM/ATR and various proteins
regulated by ATM/ATR. Particularly, the AIM3 protein shows the
activity of inducing the expression of p53, one of the
ATM/ATR-regulated proteins, and the expression of its target genes,
so as to stimulate the apoptosis of cells for DNA damage and to
inhibit the proliferation of tumor cells. Such characteristics of
AIM3 may be used to screen a substance effective for
treating/preventing ATM/ATR-mediated diseases, particularly cancer.
Accordingly, the present invention provides a method for screening
a substance effective for treating or preventing ATM/ATR-mediated
diseases, which comprise the step of: (a) culturing the AIM3
protein or a recombinant cell expressing the AIM3 protein together
with a candidate substance; and (b) determining the effect of the
candidate substance on an increase in the activity of AIM3 or the
intracellular level thereof. As used herein, the term "activity of
the AIM3 protein" refers to the binding activity with ATM/ATR, the
activity of promoting the phosphorylation of ATM/ATR or proteins
regulated by ATM/ATR, and/or the activity of inducing the
expression of p53 and its target genes. The term "increase in the
intracellular level of the AIM3 protein" means the increase in the
concentration of the AIM3 protein by the increase of expression of
the AIM3 gene or the inhibition of the degradation of the AIM3
proteins.
[0070] The expression of the AIM3 gene includes process for the
transcription of the AIM3 gene and the translation into proteins.
Accordingly, the substances screened in the present invention has
the property of: promoting the binding of AIM3 to ATM/ATR;
activating ATM/ATR or proteins regulated by ATM/ATR; inducing the
expression of p53 and its target genes; and/or increasing the
intracellular level of the AIM3 protein. These substances include
not only proteins but also naturally occurring or chemically
synthesized compounds or extracts.
[0071] The activity and intracellular level of the AIM3 protein can
be measured by various methods known in the art. Exemplary methods
include, but are not limited to, co-immunoprecipitation,
enzyme-linked immunosorbent assay, radioimmunoassay (RIA),
immunohistochemical assay, Western blotting, and fluorescence
activated cell sorter (FACS) analysis.
[0072] In addition, for the screening method using the AIM 3 of the
invention as a target gene, high throughput screening (HTS) can be
applied. The HTS is a method for screening the biological
activities of a number of candidate substances simultaneously or
almost simultaneously by testing the candidate substances
simultaneously. In a certain embodiment, cell lines are cultured in
a 96-well microtiter plate or a 192-well microtiter plate, into
which a number of candidate substances are added and then measured
for the expression of AIM3 by an immunohistochemical method. In
this format, 96 independent tests may be simultaneously performed
in a single 8 cm.times.12 cm plastic plate containing 96 reaction
wells. The wells require an assay volume of 50-500 .mu.l typically.
In addition to the plate, a number of gauges, instruments,
pipetters, robots, plate washers and plate readers are commercially
available in order to make the 96-well format suitable for a wide
range of homogeneous and heterogeneous assays.
[0073] Meanwhile, the expression level of the AIM3 gene or protein
in biological samples (e.g., blood, serum, sputum, urine and/or
tumor biopsies) collected from subjects can be compared with that
of normal persons so as to diagnose (identify) subjects having the
risk of ATM/ATR-mediated diseases. Specifically, using one selected
from the group consisting of an AIM3 protein-encoding nucleic acid,
a fragment thereof, a peptides encoded by them, and an antibody to
the peptide as a primer or probe, ATM/ATR-mediated diseases may be
identified. Accordingly, the present invention provides a method
for identifying a subject having the risk of ATM/ATR-mediated
diseases, which comprise the steps of: (a) measuring the expression
level of AIM3 in a tissue sampled from a subject; and (b) comparing
the level of AIM3 in the tissue with a normal AIM3 level. The
methods for identifying such a disease include those which are
capable of detecting the expression of AIM3 at a transcriptional or
translational level (such as RT-PCR, Northern blotting, Western
blotting, immunological assays and so on). This method is very
effective for diagnosing cancer among ATM/ATR-mediated
diseases.
[0074] In still another aspect, the present invention provides a
kit for the diagnosis of ATM/ATR-mediated diseases, which comprises
one selected from the group consisting of a AIM3-encoding nucleic
acid, a fragment thereof, a peptide encoded by them, and an
antibody to the peptide. The AIM3 protein-encoding nucleic acid and
a fragment thereof may be synthesized with reference to the known
sequence of the AIM3 gene. The fragment of nucleic acid is
preferably a primer capable of amplifying the AIM3 gene. The
peptide encoded by the AIM3 protein-encoding nucleic acid or its
fragment may be synthesized by any technique known in the art
(Creighton, Proteins: Structures and Molecular Principles, W.H.
Freeman and Co., NY, 1983). The peptide can be produced by the
conventional stepwise liquid or solid phase synthesis, fragment
condensation, F-MOC or T-BOC chemistry (Williams et al., Eds.,
Chemical Approaches to the Synthesis of Peptides and Proteins, CRC
Press, Boca Raton Fla., 1997; Atherton .quadrature. Sheppard, Eds.,
A Practical Approach, IRL Press, Oxford, England, 1989).
[0075] The antibody to the peptide can be produced using the AIM3
protein or its fragment as an antigen by any conventional method
widely known in the immunological field. The antibodies include
polyclonal antibodies and monoclonal antibodies.
[0076] The polyclonal antibodies can be prepared from a variety of
warm-blooded animals, such as horses, cattle, goats, sheep, dogs,
fowl, turkeys, rabbits, mice or rats, by any conventional technique
known in the art. Namely, the animals are immunized by
intraperitoneal, intramuscular, intraocular or subcutaneous
injection of an antigen. The immunogenicity to the antigen can be
increased by the use of an adjuvant, for example Freund's complete
adjuvant or incomplete adjuvant. Following booster immunization, a
small serum sample was collected and tested for the reactivity to
the target antigen. Once the animal's titer reaches a stagnant
state in view of its reactivity to the antigen, a large amount of
the polyclonal antibodies can be obtained by bleeding the animal at
one-week intervals or by blood-letting the animal.
[0077] The monoclonal antibodies can also be produced by a known
method (Kennettm McKearn, and Bechtol(eds.), Monoclonal Antibodies,
Hybridomas; A New Dimension in Biological Analyses, Plenum Press,
1980). The monoclonal antibodies can be produced by immunizing an
animal with the AIM3 protein or its fragment as an immunogen,
fusing the splenocytes of the immunized animal with myeloma cells
to produce a hybridomas, screening a hybridoma that selectively
recognizes the AIM3 protein, culturing the screened hybridoma, and
isolating antibodies from the hybridoma culture. Alternately, the
monoclonal antibodies according to the present invention may also
be prepared by injecting said hybridoma into an animal, and after a
given period of time, isolating antibodies from the collected
ascites of the animal.
[0078] The antibody contained in the inventive diagnostic kit is
preferably immobilized onto a solid substrate. The antibody can be
immobilized by various techniques described in literatures
(Antibodies: A Labotory Manual, Harlow .quadrature. Lane; Cold
SpringHarbor, 1988). Suitable solid substrates include those
supported by rods, synthetic glass, agarose beads, cups, flat
packs, or other solid support or those having a film or coating
attached to them. In addition, other solid substrates include cell
culture plates, ELISA plates, tubes and polymeric films.
[0079] The diagnostic kit according to the present invention may
contain, in addition to an antibody selectively recognizing the
AIM3 protein, reagents which are used in immunological assays. The
immunological assays may include methods capable of measuring the
binding of an antigen to the antibody of the present invention.
These methods are known in the art and include, for example,
immunocytochemical assays, immunohistochemical assays,
radioimmunoassays, ELISA (enzyme linked immunoabsorbent assay),
immunoblotting, Farr assays, precipitin reaction, turbidimetry,
immunodiffusion, counter-current electrophoresis, single radical
immunodiffusion and immunofluorescence.
[0080] Reagents which are used in the immunological assays include
a suitable carrier, a labeling substance capable of emitting
detectable signals, a solubilizer and a washing agent. Furthermore,
if the labeling substance is enzyme, a substrate capable of
measuring enzymatic activity and a reaction stopping agent may be
used.
[0081] Suitable carriers include, but are not limited to, soluble
carriers, for example, one of biologically acceptable buffers known
in the art (e.g., PBS), insoluble carriers, for example
polystyrene, polyethylene, polypropylene, polyester,
polyacrylonitrile, fluorine resin, crosslinked dextran,
polysaccharide, polymers, such as latex containing magnetic fine
particles plated with metal, paper, glass, metal, agarose and
combinations thereof.
[0082] Labeling substances capable of emitting detectable signals
include enzymes, fluorescent substances, luminescent substances and
radioactive substances. The enzymes include peroxidase, alkaline
phosphatase, .beta.-D-galactosidase, glycose oxidase, maleate
dihydrogenase, glucose-6-phosphodihydrogenase, invertase and so on.
The fluorescent substances include fluorescein isothiocyanate and
phycobili-protein. As luminescent substances, isolucinol and
lucigenin and so on can be used. And, as radioactive substances,
I.sup.131, C.sup.14, H.sup.3 and so on, can be used. However, the
above examples are only examples and anything used in immunoassay
can be used. The ATM/ATR-mediated diseases which can be diagnosed
with the inventive kit are the same as described above, and
preferably, may be lung cancer, colon cancer, liver cancer,
lymphoma and leukemia.
[0083] In one embodiment of the present invention, in order to
identify the biological functions of AIM3, AIM3 gene-deficient mice
were produced by a gene trap method. Then, genomic DNA mutated by
the insertion of a gene trap vector was introduced into the
embryonic stem cell of the mice so as to construct a mutant
library. Clones containing the mutated AIM3 gene were searched from
the library and used to prepare AIM3 heterozygous mutant mice.
[0084] In another embodiment of the present invention, the sequence
of an AIM3 allele in the mutant mice was analyzed. The results
showed that the gene trap vector was integrated between the first
and second exons in the AIM3 gene (see FIG. 1a). Furthermore,
genomic PCR and Southern blotting were performed to determine AIM3
mutation (FIGS. 1b and 1c), and the expression level of AIM3 by the
mutation was determined using Western blot (see FIG. 1d).
[0085] The post-natal genotype of progenies obtained by
crossbreeding the AIM3 heterozygous mutant mice (hereinafter,
referred to as "AIM3.sup..+-. mice") and the genotype of embryos
with the passage of time were examined and the results showed that
the AIM3.sup..+-. mice appeared at a similar ratio to that of
wild-type littermates (see Table 1). This indicates that about 50%
of the AIM3.sup..+-. mice were dead in the pre-natal stage. AIM3
homozygous mice (hereinafter, referred to as "AIM3.sup.-/- mice")
would die during the early embryonic stage (see Tables 1 and 2).
This suggests that AIM3 performs an important role in vivo.
Particularly, considering that the genetic eradication of proteins,
such as Rad51, Chk1/2 and ATR involved in the DNA-damaging response
and repair system, causes early embryonic lethality (de Klein et
al., Curr. Biol., 10:479-482, 2000; Lim and Hasty, Mol. Cell Biol.,
14:7133-7143, 1996; Takai et al., Genes Dev., 14:1439-1447, 2000),
it was believed that AIM3 would be involved in the DNA-damaging
response and repair system.
[0086] Since the AIM3 protein is related to a multi-tRNA synthetase
complex involved in protein synthesis (Han et al., Biochem.
Biophys. Res. Commun., 303:985-993, 2003), the inventors expected
that a reduction of the AIM3 level would have an effect on the
overall body growth of mice. However, it was interestingly shown
that the growth rate of the AIM3.sup..+-. mice was similar to or
slightly higher than that of wild-type mice regardless of their sex
(data not shown). This suggests that protein synthesis is not
inhibited by a reduction of the AIM3 level.
[0087] In another embodiment of the present invention, we examined
the histological characteristics of tissues and organs isolated
from the AIM3.sup..+-. mice in order to identify the function of
the AIM3 gene. The results showed that various tumors were found in
the tissues and organs isolated from the AIM3.sup..+-. mice, and
the incidence of the tumors was significantly increased when their
age passed over 15 months (see FIGS. 2a to 2c and Table 3).
Particularly, in the AIM3.sup..+-. mice, lymphomas have developed
at a high frequency (see Table 3). This is consistent with the
previous report that the loss of DNA repair functions can evoke
lymphomas (Bassing et al., Cell, 114:359-370, 2003; Celeste et al.,
Cell, 114:371-383, 2003). From the result that various tumors
spontaneously formed in the AIM3-deficient mice, it is suggested
that AIM3 is a powerful tumor suppressor involved in the
tumorigenic pathway.
[0088] A rapid cell cycle is a typical indication for tumorigenesis
(Evan and Vousden, Nature, 411:342-348, 2001). Thus, in another
embodiment of the present invention, we examined whether AIM3 is
involved in cell cycle control. As a result, cells isolated from
the AIM3-deficient mice increased faster than those of wild-type
mice and showed faster cell cycle (see FIGS. 3a and 3b).
Furthermore, the expression of AIM3 in cell cycles was examined by
Western blot analysis and flow cytometry, and the results showed
that AIM3 was significantly induced during DNA synthetic phase (see
FIGS. 3c and 3d). To understand the functional reason for the AIM3
induction during DNA synthetic phase, the cellular localization of
AIM3 in growth arrest state and proliferation condition was
examined. According to the results, AIM3 was detected mainly in
cytoplasm when the cell growth was suppressed by serum starvation.
However, it was detected in nucleus when the cells resumed growth
(see FIG. 3e). This indicates that, during DNA synthesis, AIM3 is
not only induced but also translocated into nuclei. Such results
suggest that AIM3 can perform novel functions within the
nuclei.
[0089] Cell responses to DNA damage include cell cycle arrest,
apoptosis, and direct activation of DNA repair networks (Zhou B B
et al., Cancer Biol. Ther., S (4 Suppl 1):S16-22, 2003). Also, the
resistance to apoptosis, one of cell responses, is a typical
indication for tumorigenesis (Evan and Vousden, Nature,
411:342-348, 2001). Thus, in order to examine whether AIM3 is
involved in apoptosis regulation, the response of AIM3.sup..+-.
mouse-derived cells was examined using adriamycin that induces DNA
damage. The AIM3.sup..+-. mouse-derived cells show the resistance
to apoptosis (see FIG. 4a). Moreover, the growth of wild-type mouse
cells was completely arrested by adriamycin, whereas that of the
AIM3.sup..+-. mouse-derived cells was slightly inhibited (see FIG.
4b). A change in the AIM3 level caused by DNA damage was examined
and the results showed that the expression of AIM3 was induced at
both transcriptional and translational levels by treatment with a
DNA-damaging agent such as adriamycin (see FIG. 4c). In addition,
the cellular localization of AIM3 caused by DNA damage was
examined. The results showed that nuclear foci formed by AIM3 were
remarkably increased in UV-irradiated cells (see FIG. 4d). All of
these results suggest that AIM3 is involved in the responses to DNA
damage induced by genotoxic stress, and it is translocated into the
nuclei when DNA is damaged.
[0090] In still another embodiment of the present invention, it was
examined whether AIM3 is involved in cell proliferation. The
results showed that cells and tissues derived from the
AIM3-deficient mice had a cell proliferation rate higher than those
of wild-type mouse cells (see FIGS. 5a and 5b). In addition, the
level of proliferation of cells transfected with the AIM3 gene was
lower than that of wild-type mouse cells (see FIG. 5c). This
suggests that AIM3 shows the anti-proliferation activity against
tumor cells.
[0091] According to the above results indicating that AIM3 is
induced at a high level during DNA synthetic phase and when DNA get
damaged, and has anti-proliferation activity similar to other DNA
repair proteins (Falck et al., Nature, 410:842-847, 2001; Lim et
al., Mol. Cell, 7:683-694, 2000), it can be found that AIM3 is
functionally involved in signal transduction pathways that respond
to the repair of DNA damage caused by the DNA replication or
stress.
[0092] Meanwhile, it is known that p53, a tumor suppressor gene,
does functions of not only inhibiting the abnormal division and
proliferation of cells but also arresting the cell cycle in the
case of cellular DNA damage so as to repair the damaged DNA, and
p53 is involved in cell proliferation and apoptosis to prevent DNA
from being unlimitedly amplified (Levine, Cell, 88:323-331, 1997;
Vousden, Cell, 103:691-694, 2000). Thus, the inventors examined the
functional connection between AIM3 and p53. The results showed
that, in cells transfected with the AIM3 gene, the levels of not
only p53 but also its target gene, p21, were increased (see FIGS.
6a and 6b). This increase in the gene level was further increased
by treatment with adriamycin that induces apoptosis (see FIG. 6c).
The level of proliferation of the AIM3 gene-transfected cells was
lower than that of wild-type cells, and anti-proliferation activity
of AIM3 was abolished in p53- or p21-deficient cancer cells (see
FIG. 6d). The induction of p53 by UV or adriamycin was blocked when
AIM3 was suppressed (see FIG. 6e). This indicates that AIM3
upregulates the expression of p53 induced by DNA damage and its
target gene p21, thus inhibiting the proliferation of cancer
cells.
[0093] It is known that mammalian ATM and ATR playing a key role in
cell cycle checkpoints initiated by DNA damage are serine-threonine
kinases which are involved in DNA repair processes responding to
other genotoxic stresses (Yang et al., Carcinogenesis, 24:
1571-1580, 2003). Furthemore, ATM and ATR not only activate
directly p53 in response to DNA damage but also regulate the cell
cycle via p53 (Abraham, Genes Dev., 15:2177-2196, 2001). Thus, in
order to examine whether AIM3 regulates p53 via ATM/ATR, the
present inventors examined whether ATM/ATR inhibitors inhibit the
activity of AIM3. The results indicated that the anti-proliferation
activity of AIM3, the apoptosis induced by AIM3, and the
AIM3-dependent expression of p53, were all inhibited by caffeine,
inhibitor of ATM/ATR (see FIGS. 7a to 7c). Moreover, the
AIM3-dependent expression of p53 was also blocked by the expression
of the kinase-dead domain of ATM (KD-ATM) that inhibits
specifically the activity of ATM (see FIG. 7d). These results
suggest that AIM3 acts through ATM/ATR.
[0094] In order to examine the relation between AIM3 and ATM/ATR in
more detail, the present inventors analyzed the interaction between
AIM3 and ATM/ATR.
[0095] The results indicated that the interaction between the AIM3
and ATM/ATR was enhanced by stresses, such as the exposure to UV,
adriamycin treatments, etc., and the interaction was done by the
specific binding of AIM3 to the FAT domain of ATM/ATR (FIGS. 8a to
8c).
[0096] Then, the present inventors examined whether the activity of
ATM/ATR is enhanced by the association with AIM3. The results
showed that the phosphorylation level of H2AX in the AIM3.sup..+-.
mouse-derived cells, which is a substrate for ATM/ATR, was
significantly lower than that of H2AX in wild-type mouse cells (see
FIG. 9a). Furthermore, the phosphorylation of H2AX was blocked by
the expression of antisense-AIM3 (As-p18) (see FIG. 9b). In
addition, the inventors found that AIM3 increased the
phosphorylation of ATM and its target proteins (p53 and chk2)
through various tests (FIG. 9c and data not shown). These results
suggest that AIM3 interacts directly with ATM/ATR to activate not
only ATM/ATR but also ATM/ATR-regulated proteins.
[0097] Finally, in order to examine the functional association
between AIM3 and ATM/ATR-mediated diseases, the present inventors
examined the expression level of AIM3 in various cancer cell lines.
The results indicated that the expression level of AIM3 was reduced
in some cancer cell lines (see FIG. 10a). To have a clue to the
possible cause for the results, the present inventors compared the
DNA content for AIM3 gene using genomic PCR analysis. As a result,
it was confirmed that some cancer cell lines appeared to contain
less amount of DNA than other cells. This indicates that the cell
lines have loss of one allele for AIM3 (see FIG. 10b). Furthermore,
we examined the expression level of AIM3 in tissues isolated from 9
leukemia patients and, as a result, found that AIM3 was expressed
at a low level in the tissues of three patients. In this case, the
expression of p21, a p53 target gene, was also strongly suppressed
(see FIG. 10c). The level of AIM3 in normal tissues and cancer
tissues isolated from liver cancer patients was analyzed by RT-PCR.
As a result, it was confirmed that the level of AIM3 in the cancer
tissues was cancer-specifically reduced (see FIG. 10d). These
results suggest that the low level expression of AIM3 is
association with various cancer cell lines and the tissues of
cancer patients at high frequency.
[0098] As described above, it was first found in the present
invention that AIM3 is a tumor suppressor gene and particularly, a
haploinsufficient tumor suppressor gene acting in signal
transduction pathways including ATM/ATR and p53.
BRIEF DESCRIPTION OF THE DRAWINGS
[0099] FIG. 1a is a schematic representation of a gene trap vector
inserted into an AIM3 gene.
[0100] FIG. 1b shows the results of genomic PCR analysis to
determine the insertion of a gene trap vector.
[0101] M: molecular weight marker
[0102] +/+: wild-type mice
[0103] .+-.: AIM3 heterozygous mice
[0104] FIG. 1c shows the results of Southern blot analysis to
determine the insertion of a gene trap vector.
[0105] +/+: wild-type mice
[0106] .+-.: AIM3 heterozygous mice
[0107] FIG. 1d shows the results of Western blot analysis to
determine the expression level of AIM3 in various organs of
wild-type mice (+/+) and AIM3 heterozygous mice (.+-.).
[0108] FIG. 2a shows the results of immunohistochemical staining of
various tissues and organs isolated from AIM3 heterozygous
mice.
[0109] FIG. 2b illustrates the results using an anti-B220
monoclonal antibody, which shows that lymphoma cells metastasized
into liver and lung.
[0110] FIG. 2c shows the results of analysis of the incidence of
tumors at different ages (months) in wild-type mice (+/+) and AIM3
heterozygous mice (.+-.).
[0111] White bar: the numbers of autopsied wild-type mice (+/+)
[0112] Gray bar: the numbers of autopsied AIM3 heterozygous mice
(.+-.)
[0113] Black section: the numbers of mice with tumors (tumor +)
[0114] FIG. 3a shows the results of cell counting to measure the
proliferation rate of the splenocytes and thymocytes isolated from
wild-type mice (+/+) and AIM3 heterozygous (.+-.).
[0115] FIG. 3b shows the results of analysis of the cell cycle of
splenocytes isolated from wild-type mice (+/+) and AIM3
heterozygous mice (.+-.).
[0116] FIG. 3c shows the results of Western blot analysis to
determine the expression level of AIM3 in each phase of the cell
cycle.
[0117] FIG. 3d shows the results of FACS analysis to determine the
expression level of AIM3 at different cell cycle.
[0118] Left panel: DNA content(Y-axis) and the expression of AIM3
(X-axis) are analyzed by FACS and the density of cell is
illustrated in contour lines. The "S" portion represents cells in
the DNA synthetic phase on the basis of DNA content, and the "G1"
portion represents cells in the G1/G0 phase.
[0119] Right panel: the expression level of AIM3 in "G1" and "S"
portion respectively in the left panel is shown in histograms. The
X-axis represents the expression level of AIM3, and the Y-axis
represents cell number.
[0120] FIG. 3e shows the results of observation of the cellular
localization of AIM3 at different proliferation conditions of
cells.
[0121] SF: cell culture in serum-free media
[0122] CM: cell culture in complete media
[0123] FIG. 4a shows the results of flow cytometry to examine the
apoptotic responses of splenocytes of AIM3 heterozygous mice (.+-.)
to adriamycin treatment(Adr), as compared to those of wild-type
mice (+/+).
[0124] M1: annexin V-FITC positive populations FIG. 4b shows the
results of flow cytometry to examine the response of wild-type
mice- and AIM3 heterozygous mice (.+-.)-derived cells to adriamycin
treatment, which caused cell growth arrest.
[0125] FIG. 4c shows the results of RT-PCR analysis and Western
blot analysis to examine changes in the expression level of AIM3 by
treatment with adriamycin (Adr), at different time.
[0126] Bars represent the population of G1/G0 phase cells and
numbers represent the percentage of G1/G0 phase cells
[0127] FIG. 4d shows the results of immunofluorescent staining to
observe the cellular localization of AIM upon exposure to UV.
[0128] FIG. 5a illustrates the results of thymidine incorporation
to measure the cell proliferation rate of mouse embryonic
fibroblasts (MEFs) isolated from wild-type mice (+/+) and AIM3
heterozygous mice (.+-.).
[0129] FIG. 5b illustrates the results of immunofluorescent
staining using an anti-Ki67 antibody (green color), to measure the
cell proliferation rate of various tissues isolated from wild-type
mice (+/+) and AIM3 heterozygous mice (.+-.).
[0130] FIG. 5c illustrates the results of thymidine incorporation
to measure the proliferation rate of cells transfected with an AIM3
gene.
[0131] EV: HCT116 cells transfected with an empty vector containing
no AIM3 gene
[0132] AIM3: HCT 116 cells transfected with an AIM3 expression
vector
[0133] FIG. 6a shows the results of Western blot analysis to
examine the effect of AIM3 on p53 expression in mouse embryonic
fibroblasts (MEFs) derived from AIM3 heterozygous mice (.+-.) and
HCT116 cells transfected with an AIM3 expression vector.
[0134] +/+: wild-type mice
[0135] .+-.: AIM3 heterozygous mice
[0136] EV: HCT116 cells transfected with an empty vector containing
no AIM3 gene
[0137] AIM3: HCT116 cells transfected with an AIM3 expression
vector
[0138] FIG. 6b shows the results of RT-PCR to examine the effect of
AIM3 on the p53-dependent transcription of p21.
[0139] -: HCT116 cells transfected with an AIM3 expression
vector
[0140] +: HCT116 cells transfected with an empty vactor containing
no AIM3 gene
[0141] FIG. 6c shows the results of luciferase assay using a vector
containing a p21 promoter-fused luciferase gene, to examine the
effect of AIM3 on the p53-dependent transcription of p21.
[0142] EV: HCT116 cells transfected with an empty vector containing
no AIM3 were not treated with anything
[0143] EV+Adr: HCT116 cells transfected with an empty vector
containing no AIM3 were treated with adriamycin
[0144] AIM3: HCT116 cells transfected with an AIM3 expression
vector were not treated with anything
[0145] AIM3+Adr: HCT116 cells transfected with an AIM3 expression
vector were treated with adriamycin
[0146] FIG. 6d illustrates the effect of ATM3 on the proliferation
of wild-type HCT116 cells (WT), p53 gene-null HCT116 cells (p53-/-)
and p21 gene-null HCT116 cells (p21-/-).
[0147] EV: HCT116 cells transfected with an empty vector containing
no AIM3 gene
[0148] AIM3: HCT116 cells transfected with an AIM3 expression
vector
[0149] FIG. 6e shows the effect of AIM3 on p53 induction caused by
exposure to UV and treatment with adriamycin (Adr).
[0150] EV: HCT116 cells transfected with an empty vector containing
no antisense-AIM3 (As-AIM3)
[0151] As-AIM3: HCT116 cells transfected with a vector containing
antisense-AIM3
[0152] FIG. 7a illustrates the effect of caffeine on the
anti-proliferation activity of AIM3.
[0153] EV: HCT116 cells transfected with an empty vector containing
no AIM3 gene
[0154] AIM3: HCT116 cells transfected with an AIM3 expression
vector
[0155] FIG. 7b shows the effect of caffeine on AIM3-induced
apoptosis.
[0156] FIG. 7c shows the results of Western blot analysis to
examine the effect of AIM3 on the induction of p53, after treatment
with caffeine, an ATM inhibitor.
[0157] -: HCT116 cells transfected with an empty vector containing
no AIM3 gene
[0158] +: HCT116 cells transfected with an AIM3 expression
vector
[0159] FIG. 7d shows the results of Western blot analysis to
examine the effect of AIM3 on the induction of p53, after
introducing a KD-ATM domain, a specific inhibitor of ATM activity,
into cells.
[0160] -: HCT116 cells transfected with an empty vector containing
no AIM3 gene
[0161] +: HCT116 cells transfected with an AIM3 expression
vector
[0162] FIG. 8a shows the results of co-immunoprecipitation to
determine the interaction between AIM3 and ATM, after treatment
with UV and adriamycin.
[0163] FIG. 8b shows the results of in vitro pull-down assay to
determine the direct interaction between AIM3 and ATM.
[0164] FIG. 8c shows the results of co-immunoprecipitation to
determine the interaction between AIM3 and ATR, after exposure to
UV.
[0165] FIG. 9a shows the results of Western blot analysis to
measure the phosphorylation level of H2AX, a substrate of ATM, in
splenocytes and thymocytes isolated from wild-type mice (+/+) and
AIM3 heterozygous mice (.+-.).
[0166] p-H2AX: phosphorylated H2AX
[0167] FIG. 9b shows the results of Western blot analysis to
examine the effect of AIM3 on the phosphorylation of H2AX, a
substrate of ATM, using antisense-AIM3 (As-AIM3).
[0168] -: no treatment with VP 16, an apoptosis-inducer
[0169] +: treatment with VP 16, an apoptosis-inducer
[0170] FIG. 9c shows the results of Western blot analysis to
examine the effect of AIM3 on the phosphorylation of ATM and its
target proteins (p53 and Chk2).
[0171] p-ATM: phosphorylated ATM
[0172] p-p53: phosphorylated p53
[0173] p-Chk2: phosphorylated Chk2
[0174] actin: loading control
[0175] FIG. 10a shows the results of RT-PCR to measure the
expression level of AIM3 in different human cancer cell lines.
[0176] FIG. 10b shows the results of genomic PCR to examine the DNA
content for AIM3 gene in different human cancer cell lines.
[0177] FIG. 10c shows the results of Western blot analysis to
measure the expression level of AIM3 and p21 in tissues isolated
from 9 leukemia patients.
[0178] APML: acute promyelocytic leukemia
[0179] CML: chronic myelocytic leukemia
[0180] FIG. 10d shows the results of RT-PCR to measure the
expression levels of AIM3 in normal tissues and cancer tissues
isolated from 9 liver cancer patients.
[0181] N: normal tissues at tumor-adjacent sites
[0182] T: liver cancer tissues
BEST MODE FOR CARRYING OUT THE INVENTION
[0183] Hereinafter, the present invention will be described in
detail by the following examples. It is to be understood, however,
that these examples are given for illustrative purpose only and are
not construed to limit the scope of the present invention.
EXAMPLE 1
[0184] Generation of AIM3 Gene-deficient Mutant Mice
[0185] The present inventors generated AIM3-deficient mice by a
gene trap method (Zambrowicz, B. P. et al., Nature, 392:608-611,
1998). Among the embryonic stem cell library of 129/SvEvBrd mouse
in which the gene trap vector was randomly introduced (OmniBank
Library, Lexicon Genetics), the OST377244 clone including AIM3
genes mutated by the integration of the gene trap vectors was found
out. Using this clone, C57BL6/albino AIM3 heterozygous mice were
generated following the standard protocol of Lexicon Genetics, Inc.
The heterozygous mice were interbred to generate the homozygous
offspring.
EXAMPLE 2
[0186] Examination of Genotypic and Phenotypic Characteristics of
AIM3 Gene-deficient Mice
[0187] <2-1> Determination of Site of Gene Trap Vector
Insertion in AIM3 Allele
[0188] The site of a gene trap vector insertion in an AIM3 mutant
allele was determined by sequencing analysis. Here, the sequencing
was performed by Pangenomics, a sequencing company. As shown in
FIG. 1a, the sequencing results indicated that the gene trap vector
was inserted between exon I and exon II of the AIM3 gene.
[0189] <2-2> Genomic PCR Analysis
[0190] From the tail of each of the mice generated in <Example
1>, genomic DNA was isolated. Then, about 1.5-kb DNA fragment
containing the exon I region of the AIM3 gene was amplified by PCR
with a primer pair of p18F-1 and p18R-1 (SEQ ID NO: 3 and SEQ ID
NO: 4) (see FIG. 1a). In addition, about 0.8-kb DNA fragment
containing a part of the AIM3 gene and a part of the gene trap
vector was amplified by PCR with the p18F-1 primer and an LTR
primer (SEQ ID NO: 5) binding to the gene trap vector (about 5.7
kb) integrated into the genome (see FIG. 1a). The PCR reaction
consisted of the following: denaturation of template DNA at
94.degree. C. for 5 min; and then, 30 cycles of 1 min at 94.degree.
C., 1 min at 54.degree. C., and 2 min at 72.degree. C.
[0191] Interestingly, all of the generated mutant mice were
heterozygote (AIM3.sup..+-. mice) producing both of 1.5 and 0.8 kb
DNA fragments (see FIG. 1b). On the other hand, in the case of
wild-type mice (AIM3.sup.+/+ mice), only the 1.5-kb band could be
found.
[0192] <2-3> Southern Blot Analysis
[0193] From the tail of each mouse, genomic DNA was isolated and
digested with SacI, followed by gel electrophoresis to separate the
digested DNA fragments. Then, a PCR product amplified with p18F-2
and p18R-2 primers shown in SEQ ID NO: 6 and SEQ ID NO: 7, which
contains the exon II region of the AIM3 gene, was labeled with a
radioactive isotope (see FIG. 1), and the labeled probe was
hybridized with the digested DNA fragments (southern, E. M., J.
Mol. Biol., 98:503, 1975).
[0194] As shown in FIG. 1c, a band of about 12 kb was detected in
wild-type mice but additional band of about 3 kb was detected in
the heterozygous mice.
[0195] <2-4> Determination of Induction of Embryonic
Lethality Caused By AIM3 Gene Deletion
[0196] In the analysis in Examples <2-2> and <2-3>,
offspring with a homozygous genotype could not be found. Thus, in
order to examine whether the deficiency of the AIM3 gene induces
embryonic lethality, the genotype of post-natal mice and the
genotype of embryos on different time after fertilization were
examined by genomic PCR according to the same method as in Example
<2-2>. The results are shown in Tables 1 and 2 below.
[0197] As shown in Table 1, among a total of 262 survival mice, 114
mice were wild type (+/+) and 148 mice were heterozygous (.+-.).
None of surviving mice was homozygous (-/-). Particularly, the
heterozygous mice were born at a similar ratio with the wild-type
littermates, indicating that about 50% of the heterozygous mice
would die during the pre-natal stage. As shown in Table 2, among
total of 38 embryos isolated at 7.5-9.5 days after fertilization,
only one embryo at 8.5 days containing the homozygous genotype was
detected. This indicates that the AIM3 homozygous mice would be
early embryonic lethal. TABLE-US-00001 TABLE 1 Post-natal
segregation ratio of genotype from the offspring generated by the
intercrosses between the C57BL6 AIM3 heterozygous mice Total +/+
+/- -/- Number of surving mice 262 114 148 0 % 100 43.5 56.5 0
[0198] TABLE-US-00002 TABLE 2 Embryonic segregation ratio of
genotype from the offspring generated by the intercrosses between
the C57BL6 AIM3 heterozygous mice Day of Gestation Total +/+ +/-
-/- Resorbed 7.5 days 28 7 15 0 6 8.5 days 34 11 16 1 6 9.5 days 21
8 8 0 5 Total 83 26 39 1 17 % 31.3 47.0 1.2 20.5
[0199] The results suggest that loss of AIM genes leads to
embryonic lethality.
[0200] <2-5> Western Blot Analysis
[0201] According to the method described in Ziak, M, et al. (Ziak,
M, et al., Biochem. Biophys. Res. Commun. 280:363-367, 2001),
proteins were isolated from various organs, such as small
intestines, kidneys, heart and spleen. Then, according to the
method described in Park S. G., et al. (Park S. G., et al., J.
Biological Chemistry 274:16673-16676, 1999), Western blot analysis
was performed using a polyclonal rabbit anti-AIM3 antibody. The
anti-AIM3 antibody was prepared according to the method described
in Kim, T. et al. aim, T. et al., J. Biol. Chem., 275:21768-21772,
2000).
[0202] As shown in FIG. 1d, although the degree of reduction varied
depending on the organs, the expression level of AIM3 in the organs
of the AIM3.sup..+-. mice was significantly lower than that in the
organs of wild-type mice.
EXAMPLE 3
[0203] Examination of Histological Characteristics of AIM3.sup..+-.
Mice
[0204] In order to determine the functions of the AIM3 gene, the
present inventors isolated tissues and organs from the
AIM3.sup..+-. mice and analyzed the histological characteristics of
the isolated tissues and organs.
[0205] At first, after sacrifing mice at given time intervals,
various tissues were isolated and fixed with 10% formalin. The
fixed tissues were embedded in paraffin, followed by subjecting
into H&E staining. In order to determine B cell metastasis,
immunohistochemical staining for surface marker B220 was performed
with paraffin slide. After de-paraffin using xylene, the slide was
incubated in a blocking buffer (1:100, 5% BSA and 0.1% Tween
20/PBS) containing an anti-B220 antibody (Santacruz Biotech.) for 2
hours. After the slide was washed with PBS, the tissues fixed to
the slide were incubated again with an avidin-conjugated secondary
antibody and DAB solution.
[0206] As a result, various tumors were found in the AIM3.sup..+-.
mice (see Table 3 and FIG. 2a). Interestingly, among 18
tumor-developing AIM3.sup..+-. mice, 14 mice contained lymphoma
which originated from the spleen or lymph node, and 5 mice had
complex tumors. Specifically, adenocarcinoma was found in the
breasts of 15-month-old AIM3.sup..+-. mice (B-63) and 23-month-old
AIM3.sup..+-. mice (B-95), adenocarcinoma in the seminal vesicles
of 19-month-old AIM3.sup..+-. mice (B-103), and hepatocarcinoma and
sarcoma of unknown origin in 22-month-old AIM3.sup..+-. mice
(B-207). All of these cancers showed the typical malignant
phenotypes, such as anaplasia and invasiveness. Furthermore,
lymphoma was found in the lymph nodes of 22-month-old AIM3.sup..+-.
mice (B-232) and well-differentiated carcinoma which originated
from the bronchiole epithelium was observed in 17-month-old
AIM3.sup..+-. mice (B-14).
[0207] It was found that some of lymphomas metastasized into other
organs, such as the liver, kidneys, lungs and salivary glands (see
FIG. 2b). The incidence of these tumors was remarkably increased
after 15 month-old (see FIG. 2c and Table 3). TABLE-US-00003 TABLE
3 Tumors found in AIM3.sup.+/- mice Mouse Age ID Tumor locus
Metastasis (month) Single B-2 Liver, dysplasia -* 8 solid B-268
Liver - 23 tumor B-63 Breast - 15 (adenocarcinoma) B-233 Seminal
vesicle - 21 (adenocarcinoma) Lymphoma B-191 Spleen Salivary gland,
25 kidneys, lungs B-262 Spleen Lungs 20 B-275 Mesenteric lymph
Liver, spleen 17 node B-148 Spleen - 24 B-264 Spleen Salivary gland
15 B-143 Cervical lymph node Liver, lungs 25 B-226 Spleen Lungs 22
B-261 Spleen Lungs 20 B-321 Spleen Liver 1 Multiple B-103 Seminal
vesicle Spleen, kidneys 19 tumor (adenocarcinoma), metastatic
lymphoma B-14 Lung adenocarcinoma, Salivary gland 17 metastatic
lymphoma B-95 Breast Salivary gland, 23 adenocarcinoma spleen
(solid tumor), metastatic lymphoma B-232 Lung adenocarcinoma - 22
(solid tumor), lymphoma (lymph node) B-207 Metastatic sarcoma
Spleen, liver, 22 (liver), hepatic lungs, salivary carconoma
(liver), gland metastatic lymphoma *negative
[0208] As shown in the above results, all of various tumors
spontaneously formed in the AIM3-deficient heterozygous mice led us
to suspect that AIM3 is a strong tumor suppressor involved in
general tumorigenic mechanisms.
EXAMPLE 4
[0209] Identification of Relation Between Cell Cycle and AIM3
[0210] A rapid cell cycle is a typical indicatin for tumorigenesis
(Evan and Vousden, Nature, 411:342-348, 2001). Accordingly, it was
addressed whether AIM3 could play a role in cell cycle control.
[0211] <4-1> Examination of Change of Cell Cycle in
AIM3.sup..+-. Mouse-derived Cells
[0212] First, the present inventors examined the cell proliferation
rate of AIM3.sup..+-. mouse-derived cells, compared to that of
wild-type mouse cells. For this purpose, from 4-week-old wild-type
mice and AIM3.sup..+-. mice, the splenocytes and thymocytes were
isolated, and the number of cell according to culture time was
counted. As shown in FIG. 3a, the results showed that the
AIM3.sup..+-. mice-derived cells proliferated faster than wild type
mice cells.
[0213] Then, in order to examine the cell cycle of the
AIM3.sup..+-. mice-derived cells, FACS analysis was performed. The
splenocytes isolated from 4-week-old wild-type mice and
AIM3.sup..+-. mice were incubated overnight. The incubated cells
were fixed with 1% PFA (paraformaldehyde) and stained with PI
(propidium iodide). FACS analysis was conducted on 20,000 cells per
sample. As shown in FIG. 3b, the splenocytes isolated from the
AIM3.sup..+-. mice showed faster cell cycle than the wild-type mice
cells.
[0214] <4-2> Examination of Change in Expression Level of
AIM3 with Change in Cell Cycle
[0215] In order to determine the functions of AIM3 during the cell
cycle, it was examined whether AIM3 is expressed depending on the
cell cycle. HCT116 cells incubated in a serum-free medium for 24
hours and then incubated them again in a serum-containing medium to
synchronize cell cycle. The expression level of AIM3 of the
synchronized cells in different time under serum-deprivation and
serum-re-fed conditions was measured by Western blot analysis. As a
result, the AIM3 was remarkably induced during the DNA synthetic
phase (see FIG. 3c).
[0216] In order to confirm this fact further, the present inventors
performed FACS analysis. HCT116 cells (Human colon adenocarcinoma
cell line) were fixed with 1% PFA and neutralized, and were
cultured with an anti-AIM3 monoclonal antibody. Then, the cells
were cultured with a FITC-conjugated anti-mouse goat IgG antibody
(Pierce). And then, the cells were co-stained with PI, followed by
FACS analysis. As a result, AIM3 was remarkably induced in the DNA
synthetic phase (see FIG. 3d). This coincides with the result of
Western blot analysis. All of these results indicate that AIM3 is
induced in the DNA synthetic phase.
[0217] <4-3> Examination of Cellular Localization of AIM3
Caused By Cell Proliferation
[0218] In order to understand the functional reason of AIM3
induction during the DNA synthetic phase, the present inventors
investigated the cellular localization of AIM3 in cell growth
arrest and cell proliferation conditions. For this purpose, DU145
cells (prostate cancer cell line) were cultured in each of a 10%
serum-containing RPMI-1640 medium (complete media (CM)) and a
serum-free media (SF), fixed with 100% Me-OH and reacted with an
anti-AIM3 monoclonal antibody. Then, they were reacted with
anti-mouse goat IgG-FITC (Pierce), and stained with PI. The
cellular localization of AIM3 was examined under a fluorescence
microscope.
[0219] As shown in FIG. 3e, when the cell growth was suppressed by
serum starvation, AIM3 was mainly located in cytoplasm, whereas,
when the cell growth was resumed, AIM3 was located in nuclei. Given
thus, it could be found that, during the DNA synthetic phase of the
cell cycle, AIM3 was not only induced but also translocated into
nuclei. These results suggest that AIM3 could have novel functions
in the nuclei.
EXAMPLE 5
[0220] Determination of Relation Between DNA Damage and AIM3
[0221] The damage of DNA by stresses and so on generally induces
apoptosis and cell cycle arrest (Zhou B B et al., Cancer Biol.
Ther., S(4 Suppl 1):S16-22, 2003). Thus, the present inventors
investigated the role of AIM3 in the response of cells to the
stress-induced apoptosis and cell growth arrest.
[0222] <5-1> Examination of Effect of AIM3 Gene Deletion on
Apoptosis Regulation
[0223] Using adriamycin that induces DNA damage, the response of
AIM3.sup..+-. mouse-derived splenocytes to pro-apoptotic stress was
examined.
[0224] First, the splenocytes were isolated from wild-type mice and
AIM3.sup..+-. mice. To induce apoptosis, the isolated splenocytes
were treated with 0.2 .mu.g/ml of adriamycin (Adr, Sigma) for 2
hours. Then, the cells were cultured with FITC-conjugated annexin V
(Roche) for 5 minutes. And then, the cells were washed with PBS and
subjected to FACS analysis under a FL-1H detector. In this
analysis, 20,000 cells per sample were used.
[0225] As shown in FIG. 4a, apoptotic cells were significantly
increased by treatment with adriamycin in the wild-type cells,
however the AIM3.sup..+-. cells showed the resistance to apoptosis
induced by adriamycin. This indicates that AIM3 is required for
sensitivity of cell to apoptosis induced by DNA damage. From this,
it can be found that AIM3 promotes apoptosis caused by DNA
damage.
[0226] <5-2> Examination of Change in Cell Growth Caused By
Apoptosis-inducer
[0227] In order to examine the importance of AIM3 in cell growth
arrest caused by adriamycin, flow cytometry was performed. First,
the thymocytes were isolated from wild-type mice and AIM3.sup..+-.
mice, and then treated with 0.2 .mu.g/ml of adriamycin (Adr, Sigma)
for 6 hours. Next, the cells were subjected to FACS analysis in the
same method as in Example <5-1>. As shown in FIG. 4b, the
growth of AIM3.sup..+-. mouse cells was slightly suppressed by
treatment with adriamycin, whereas that of wild-type mouse cells
was arrested.
[0228] <5-3> Examination of Change in AIM3 Level Caused By
Apoptosis-inducer
[0229] It was examined by RT-PCR analysis and Western blot analysis
whether the level of AIM3 is affected by treatment with
adriamycin.
[0230] For this purpose, HCT116 cells were treated with 0.2
.mu.g/ml of adriamycin. Then, the cells were collected at different
time and dissolved in Sol D solution (4 M guanidine thiocyanate, 1%
laurosarcosine, 25 mM sodium citrate, and 0.1% b-mercaptoethanol).
The cell extracts were incubated in acidic phenol and chloroform
containing 4% isoamylalcohol, and vortexed. The mixture was
centrifuged at 14,000 rpm. The upper layer was collected and added
with isopropanol so as to precipitate RNA. The precipitated RNA was
washed with 100% ethanol, and 1 .mu.g of RNA was dissolved in
distilled water and used as a template for RT-PCR. Then, RT-PCR was
performed with primers shown in SEQ ID NO: 8 and SEQ ID NO: 9. The
expression level of GADPH was also measured in order to
quantitatively compare that of AIM3.
[0231] Meanwhile, for Western blot analysis, cells treated with
adriamycin were dissolved in RIPA containing protease cocktail. The
solution was centrifuged at 14,000 rpm for 30 minutes. 20 .mu.g of
the extracted proteins were separated by SDS-PAGE. Then, according
to the method described in Park S. G., et al. (Park S. G., et al.,
J. Biol. Chem., 274:16673-16676, 1999), Western blot analysis was
performed using a polyclonal rabbit anti-AIM3 antibody. The
expression level of tubulin was also measured in order to
quantitatively compare that of AIM3.
[0232] As shown in FIG. 4c, both the transcription and translation
of AIM3 were induced in response to adriamycin. Moreover, the
induction of AIM3 was also observed by other DNA-damaging agents,
such as UV, actinomycin D (Act.D) and cisplatin (CDPP) (data not
shown). Particularly, AIM3 was induced within 5-10 minutes after
exposure to UV or adriamycin (data not shown). These results
indicate that AIM3 is functionally involved in signal transduction
pathways which respond to DNA repair caused by DNA replication or
DNA damage.
[0233] <5-4> Cellular Localization of AIM3 Upon DNA
Damage
[0234] The cellular localization of AIM3 upon DNA damage was
examined using U2OS cells containing large nuclei. The U2OS cells
(osteosarcoma cell line) were treated with 254-nM wavelength UV-C
(UV cross linker) at 50 J/m.sup.2. The cells were cultured in a
complete medium for 30 minutes and collected. Then, the same method
as in Example <4-3> was performed so as to examine the
cellular localization of AIM3 by immunofluorescent staining. As a
result, as shown in FIG. 4d, the UV-irradiated cells showed a
remarkable increase in nuclear foci formed by AIM3.
[0235] All of these results indicate that AIM3 is involved in
responses to DNA damage induced by genotoxic stress.
EXAMPLE 6
[0236] Identification of Relation Between Cell Proliferation and
AIM3
[0237] The present inventors found in
EXAMPLE 4> that loss of AIM3 made cell cycle faster and AIM3 was
highly induced in the DNA synthetic phase. Thus, it was examined
whether AIM3 is also involved in cell proliferation.
[0238] <6-1> Examination of Change in Cell Proliferation
Caused By Deletion of AIM3 Gene
[0239] a) Thymidine Incorporation
[0240] Mouse embryonic fibroblasts (MEFs, E14.5d) isolated from
wild-type mice and AIM3.sup..+-. mice were cultured in a medium
containing 1 .mu.Ci/ml [.sup.3H] thymine. The cultured cells were
washed with cold PBS and incubated in 10% TC A solution for 30
minutes so as to precipitate nucleic acids. Then, the cells were
dissolved in 0.1 N NaOH, and the amount of radioactive thymidine
incorporated in the precipitate was quantified by a liquid
scintillation counter. The experiments were repeated three times
and the data were averaged.
[0241] As a result, as shown in FIG. 5a, MEFs isolated from the
AIM3.sup..+-. mice had a higher proliferation rate than the
wild-type MEFs.
[0242] b) In Situ Immunofluorescence Staining
[0243] From AIM3.sup..+-. mice, the intestines, testes, spleens and
thymuses were isolated. Then, to examine the cell proliferation
rate of the isolated tissues, in situ immunofluorescence staining
was performed using Ki-67, cell proliferation marker (Gerdes J. et
al., J. Immunol., 133:1710-1715, 1984).
[0244] As a result, as shown in FIG. 5b, the proliferation of cells
in the AIM3.sup..+-. mouse-derived tissues was higher than that in
the wild-type mouse-derived tissues.
[0245] <6-2> Examination of Change in Cell Proliferation Rate
With Increase in AIM3 Expression
[0246] The present inventors found in Example <6-1> that a
reduction in the expression of AIM3 resulted in an increase in cell
proliferation. Thus, it was examined whether an increase in the
expression of AIM3 results in the suppression of cell
proliferation.
[0247] The AIM3 gene (SEQ ID NO: 2) was inserted into a pcDNA3
(Invitrogen) vector so as to prepare an AIM3 expression vector.
Then, the expression vector was transfected into HCT116 cells
(human colon adenocarcinoma cell line). The cell proliferation rate
of the transfected cells was examined in the same method as in the
part a) of Example <6-1>. As a control group, HCT116 cells
transfected with pcDNA3 vector containing no AIM3 gene (empty
vector; EV) were also used.
[0248] As a result, as shown in FIG. 5c, proliferation of cells was
reduced in the cells introduced with the AIM3 gene. This suggests
that AIM3 shows anti-proliferation activity against tumor
cells.
EXAMPLE 7
[0249] Identification of Function of AIM3 as Upregulator of p53
[0250] Tumor suppressor protein p53 plays a major role in
regulation of DNA damage-induced cell cycle arrest and apoptosis
(Levine, Cell, 88:323-331, 1997; Vousden, Cell, 103:691-694, 2000).
Thus, the functional relation between AIM3 and p53 was
examined.
[0251] <7-1> Measurement of p53 Level Caused By AIM3
[0252] The expression levels of p53 and AIM3 in mouse embryonic
fibroblasts (MEFs) isolated from AIM3.sup..+-. mice and wild-type
mice were measured with Western blot analysis according to the same
method as in Example <5-3>. Also, the expression levels of
AIM3 and p53 in the transfected HCT116 were measured with Western
blot analysis after the AIM3 expression vector prepared in Example
<6-2> was transfected into HCT116 cells.
[0253] As a result, as shown in FIG. 6a, the expression level of
p53 in the MEFs of the AIM3.sup..+-. mice was lower than that in
the MEFs of the wild-type mice. Meanwhile, the level of p53 in the
HCT116 cells transfeted with the AIM3 gene was increased as
compared to that in a control group cells transfected with an empty
vector containing no AIM3 gene. This indicates that the ectopic
expression of AIM3 elevates the expression of p53.
[0254] <7-2> Measurement of p21 Level Caused by AIM3
[0255] In order to determine whether the increase of AIM3 would
enhance the p53-dependent transcription, the AIM3-dependent
transcription of p21 known as a target gene of p53 was
examined.
[0256] The HCT116 cells transfected with the AIM3 gene (1 .mu.g/ml)
in Example <7-1> were cultured for 24 hours. Then, RT-PCR
analysis was performed in the same method as in Example
<5-3>. As a result, as shown in FIG. 6b, the expression of
p21 in the HCT116 cells transfected with the AIM3 gene was
enhanced.
[0257] <7-3> Measurement of p21 Level Caused by AIM3 and
Adriamycin
[0258] Thereafter, in order to examine the effect of AIM3 and/or
adriamycin on the transcription of p21, luciferase assay was
performed using a vector containing a p21 promoter fused to
luciferase gene.
[0259] HCT116 cells were co-transfected with a pGL-3 vector
(Promega) engineered that the luciferase gene would be expressed
under p21 promoter, and a recombinant AIM3 expression vector(1.2
.mu.g/ml) containing the AIM3 gene. Also, control group cells were
co-transfected with the pGL-3 vector and an empty vector containing
no AIM3 gene. Then, the transfected cells of each group were
treated with 0.2 .mu.g/ml of adriamycin for 2 hours. After cells
were lyzed, the cell extract were incubated with substrate of
luciferase for 30 minutes at room temperature. 5 .mu.l of each
sample was transferred to luminometer plate and luciferase activity
was measured following the manufacturer's protocol (Promega).
[0260] As a result, as shown in FIG. 6c, the luciferase activity
regulated by the p21 promoter was highly increased by transfection
with AIM3 and the luciferase activity was further increased by the
additional treatment with adriamycin.
[0261] <7-4> Identification of Relation Between
Anti-proliferation Activity of AIM3 and p53 and p21
[0262] Examples <7-1> and <7-2> demonstrated that the
expressions of p53 and p21 depend on AIM3. Thus, it was examined
whether AIM3 suppresses the proliferation of tumor cells via p53
and p21.
[0263] The AIM3 expression vector or empty vector (2
.quadrature./.quadrature.) prepared in Example <6-2> was
transfected into each of HCT116 cells (human colon adenocarcinoma
cell line), p53-null HCT116 cells and p21-null HCT116 cells. Then,
the proliferation rate of each of the transfected cells was
examined according to the same method as in the part a) of Example
<6-1>.
[0264] As a result, as shown in FIG. 6d, the anti-proliferation
activity of AIM3 was abolished by the absence of functional p53 and
p21. This indicates that AIM3 suppresses the proliferation of tumor
cells via p53 and p21.
[0265] <7-5> Measurement of Reduction in p53 Level Caused By
Inhibition of AIM3 Expression
[0266] The present inventors inhibited the expression of AIM3 by
the use of antisense-AIM3 (As-AIM3) and then examined if the
induction of p53 is influenced by the inhibition of the AIM3
expression.
[0267] First, using primers shown in SEQ ID NO: 10 and SEQ ID NO:
11, the N-terminal 176-bp region of the ATG-containing AIM3 gene
was amplified by PCR. The PCR product was inserted into a pcDNA 3.1
vector in reverse orientation. 2 .mu.g/ml of a vector containing
antisense-AIM3 was transfected into HCT116 cells. The transfected
cells were cultured for 24 hours. Then, the cells were treated with
UV and 0.2 .mu.g/ml of adriamycin, respectively. Next, using an
anti-AIM3 antibody or an anti-p53 antibody (Santacruz), Western
blot analysis was performed in the same method as in Example
<2-5>. At this time, the expression level of actin was also
measured in order to quantitatively compare the expression level of
AIM3 and p53.
[0268] As a result, as shown in FIG. 6e, the level of p53 was
increased by treatment with UV or adriamycin, whereas the
suppression of AIM3 by As-AIM3 inhibited the induction of p53. This
indicates that AIM3 is required for increasing the expression of
p53. Moreover, the transcription of PUMA, an immediate early target
gene of p53, was also increased by irradiation with WV, and its
induction was blocked when AIM3 was suppressed by As-AIM3 (data not
shown).
[0269] These results indicate that AIM3 is an important upregulator
of p53 that mediates the induction of p53 caused by DNA damage.
EXAMPLE 8
[0270] Determination of Mechanism of AIM3
[0271] ATM/ATR are substances directly activating p53 in response
to DNA damage (Canman et al., Science, 281:1677-1679, 1998; Banin S
et al., Science, 11;281 (5383):1674-7, 1998). Thus, the present
inventors examined whether AIM3 acts via ATM/ATR.
[0272] <8-1> Analysis of Caffeine-induced Inhibition of
Anti-proliferation Activity of AIM3
[0273] In order to explore the possibility, that AIM3 can regulate
p53 via ATM/ATR, the present inventors first checked the
anti-proliferation activity of AIM3 in the presence of caffeine
known as an inhibitor of ATM/ATR. HCT116 cells were transfected
with each of the AIM3 expression vector and the empty vector(2
.mu.g/ml, respectively) for 24 hours. Then, the cells were added
with 20 mM caffeine and cultured for 4 hours. Control group cells
were added with PBS. The cell proliferation rate of the cells of
each group was examined according to the same method as in the part
a) of Example <6-1>. As a result, as shown in FIG. 7a, the
anti-proliferation activity of AIM3 was abolished by caffeine, an
inhibitor of ATM/ATR. This demonstrates that AIM3 has
anti-proliferation activity via ATM/ATR.
[0274] <8-2> Analysis of Caffeine-induced Inhibition of
Apoptosis Induced by AIM3
[0275] Thereafter, the present inventors checked whether
AIM3-induced apoptosis is inhibited by caffeine, an inhibitor of
ATM/ATR. HCT116 cells were transfected with each of the AIM3
expression vector or the empty vector(4 .mu.g/ml, respectively) for
24 hours. Then, the cells were added with 20 mM caffeine and
cultured for 12 hours. Control group cells were added with PBS.
After staining the cells with PI, we checked apoptosis with
measuring for the portion (%) of sub-G1 cells. As a result, as
shown in FIG. 7b, apoptosis was induced by the expression of AIM3,
and this effect was relieved by treatment with caffeine.
[0276] <8-3> Analysis of Caffeine-induced Inhibition of
AIM3-dependent p53 Induction
[0277] Thereafter, the present inventors examined whether the
AIM3-induced expression of p53 is inhibited by caffeine. First,
HCT116 cells were transfected with each of the AIM3 expression
vector and the empty vector(2 .mu.g/ml, respectively) for 24 hours.
Then, the cells were added with 20 mM caffeine and cultured for 12
hours. Control group cells were added with PBS and cultured. Then,
in order to examine the levels of AIM3 and p53, Western blot
analysis was performed in the same method as in Example
<7-5>. At this time, the expression level of actin was also
measured in order to quantitatively compare the expression levels
of AIM3 and p53.
[0278] As a result, as shown in FIG. 7c, the AIM3-induced
expression of p53 was suppressed by caffeine. Moreover, the
expression of PUMA, a target gene of p53, was also induced by AIM3,
however it was suppressed by caffeine (data not shown). These
results indicate that ATM/ATR play an important role in the
AIM3-dependent induction of p53.
[0279] <8-4> Analysis of KD-ATM-induced Inhibition of
AIM3-dependent p53 Induction
[0280] In order to more specifically determine that ATM/ATR play an
important role in the AIM3-dependent induction of p53, HCT116 cells
were transfected with the kinase-dead domain of ATM (KD-ATM)
(Canman et al., Science, 281: 1677-1679, 1998). The KD-ATM
suppresses specifically the activity of ATM.
[0281] First, each of vectors containing the KD-ATM domain or
wild-type ATM respectively (provided by Micheal Kastan, St. Jude
Children's Hospital), was introduced into HCT116 cells with the
AIM3 expression vector(2 .mu.g/ml). Also, as a control group for
the AIM3 expression, each of these vectors was introduced into
HCT116 with the empty vector containing no AIM3 gene. Then, the
expression levels of p53 and AIM3 in the cells of each group were
examined by Western blot analysis according to the same method as
in Example <6-5>. At this time, the expression level of actin
was also measured in order to quantitatively compare the expression
levels of AIM3 and p53.
[0282] As a result, as shown in FIG. 7d, the p53 induction caused
by an increase in the AIM3 expression was blocked by KD-ATM,
whereas not by the wild-type ATM. These results further support
that ATM is required for the AIM3-dependent induction of p53.
[0283] All of these results confirm that AIM3 has
anti-proliferation activity, apoptosis-inducing activity and
p53-upregulating activity, via ATM/ATR.
EXAMPLE 9
[0284] Analysis of ATM/ATR Activation Caused By AIM3
[0285] <9-1> Analysis of Interaction Between AIM3 and ATM
[0286] a) Co-immunoprecipitation
[0287] In order to examine the interaction between AIM3 and ATM,
co-immunoprecipitation was performed. First, from HCT116 cells
treated with each of UV and 0.2 .mu.g/ml adriamycin, proteins were
extracted at different times. The protein extracts were incubated
with normal IgG and protein A/G-agarose for 2 hours and centrifuged
to remove nonspecific IgG binding proteins. After centrifugation,
the supernatant was taken, added with 2 .mu.g of an anti-ATM
antibody (Santacruz) and incubated at 4.degree. C. for 2 hours with
agitation. And then, protein A/G-agarose was added. After washing
twice with cold PBS and once with PIRA, the precipitates were
dissolved in an SDS-sample buffer, and separated by 6% SDS-PAGE.
The proteins separated by the SDS-PAGE were transferred to a PVDF
membrane, followed by reacted orderly with an anti-AIM3 single
antibody and a horseradish peroxidase conjugated secondary
antibody.
[0288] As a result, as shown in FIG. 8a, the interaction between
AIM3 and ATM was increased within 5 minutes in response to UV and
adriamycin. The dissociation kinetics of AIM3 appeared to be much
slower in adriamycin-treated cells possibly because adriamycin is
present in the media throughout the cultivation while UV stress
would affect the cells only temporarily.
[0289] b) In Vitro Pull Down Assay
[0290] To examine the direct interaction between AIM3 and ATM, GST
full-down assay was performed.
[0291] First, AIM3 was expressed as GST fusion protein and purified
according to the manufacturer's protocol (Pharmacia). Meanwhile,
since it was difficult to synthesize the whole ATM due to its large
size, the present inventors tested the interaction between the
functional domain of ATM and AIM3. For this purpose, a fragment
consisting of 612 amino acids, including the FAT domain of an ATM
structure, was amplified by PCR with primers shown in SEQ ID NO: 12
and SEQ ID NO: 13. Also, a fragment (control group) consisting of
145 amino acids, including the C-terminal domain, was amplified by
PCR with primers shown in SEQ ID NO: 14 and SEQ ID NO: 15. Then,
the amplified PCR products were subdloned into pcDNA3.1
(Invitrogen), a vector suitable for in vitro transcription and
transition. At this time, the protein was synthesized by in vitro
translation in the presence of radioactive methionine. 10 .mu.l of
the synthesized TNT product was incubated with the GST- or GST-AIM3
fusion protein-immobilized glutathion-sepharose beads for 5
minutes. Then, the beads were washed six times with a binding
buffer (PBS containing 0.2% sarcosine and 0.2% Triton X100), and
dissolved in 10% SDS-PAGE. The binding of the GST-fused AIM3 to
each domain was determined by autoradiography.
[0292] As a result, as shown in FIG. 8b, the GST-fused AIM3 protein
bound to the FAT domain, a functional domain, but not to the
C-terminal domain of ATM. This suggests that AIM3 interacts
directly with ATR.
[0293] <9-2> Analysis of Interaction Between AIM3 and ATR
[0294] The FAT domain is found in not only ATM but also ATR
(Abraham R, Genes Dev., 15:2177, 2001). Thus, the interaction
between AIM3 and ATR was tested by co-immunoprecipitation.
[0295] First, the 293 cell was transfected with an ATR vector
(provided by Elledge S., Harvard University) containing flag-tagged
ATR. The transfected 293 cell was treated with UV, from which
proteins were extracted at different time. Next, the same method as
the part a) of Example <9-1> was performed except that an
anti-FLAG antibody (Sigma) was used in place of the anti-ATM
antibody.
[0296] As a result, as shown in FIG. 8c, AIM3 was
co-immunoprecipitated with the flag-tagged ATR, and this
interaction was further enhanced upon exposure to UV. This suggests
that AIM3 also interacts with ATR as it acts on ATM.
[0297] Accordingly, it could be found from the above results that
AIM3 interacts directly with ATR/ATM.
[0298] <9-3> Analysis of ATM/ATR Activation by AIM3
[0299] The present inventors examined whether the activity of
ATM/ATR is enhanced by the association with AIM3.
[0300] a) Measurement of Phosphorylation Level of H2AX in
AIM3.sup..+-. Mouse-derived Cells
[0301] The activity of ATM/ATR was examined using H2AX known as a
substrate of ATM/ATR (Burma et al., J. Biol. Chem., 276:
42462-42467, 2001; Ward, I. M. et al., J. Biol. Chem., 276:
47759-47762, 2001; Irene M. Ward et al., J. Biol. Chem.,
279(11):9677-9680, 2004).
[0302] After isolating splenocytes and thymocytes from wild-type
mice and AIM3.sup..+-. mice, the phosphorylation level of H2AX in
the isolated cells was measured by Western blot analysis in the
same method as in Example <7-5>. As a result, as shown in
FIG. 9a, the phosphorylation of H2AX (p-H2AX) was significantly
reduced in the AIM3.sup..+-. mice-derived cells.
[0303] b) Analysis of H2AX Phosphorylation Inhibition Caused By
Antisense AIM3
[0304] Thereafter, the present inventors treated cells with VP16, a
DNA-damaging agent (Clarke et al., Nature, 362:849-852, 1993), and
examined whether the phosphorylation of H2AX is inhibited by AIM3
inhibition in the presence of antisense-ATM3 (As-AIM3). Antisense
AIM3-containing vector(2 .mu.g/ml) prepared in Example <7-5>
was introduced into HCT116 cells. The transfected cells were
cultured for 24 hours and then treated with 100 .mu.M of VP16
(Sigma), an apoptosis-inducing agent, for 4 hours. Next, using each
of an anti-53 antibody, an anti-AIM3 antibody and an anti-p-H2AX
antibody (Cell signaling), Western blot analysis was performed in
the same method as in Example <7-5>. At this time, the
expression level of actin was also measured in order to
quantitatively compare that of each protein. As a result, as shown
in FIG. 9b, the phosphorylation of H2AX was enhanced by treatment
with VP16, but inhibited by the expression of antisense-AIM3.
[0305] c) Analysis of Effect of AIM3 on ATM Activation
[0306] In order to analyze the effect of AIM3 on the
autophosphorylation of ATM, the present inventors examined the
phosphorylation of ATM and its target proteins, p53 and chk2, by
Western blot analysis. Cells isolated from wild-type mice and
AIM3.sup..+-. mice were treated with 0.2 .mu.g/ml of adriamycin.
Then, using each of an anti-phospho-serine antibody of ATM, an
anti-p53 antibody and an anti-chk2 antibody, Western blot analysis
was performed (Bakkenist and Kastan, Nature, 421:499-506, 2003). As
a result, as shown in 9c, the phosphorylation of ATM and its target
proteins in the wild-type cells was enhanced by treatment with
adriamycin, whereas that in the AIM3.sup..+-. cells was
inhibited.
[0307] All of these results indicate that AIM3 is required for the
activation of ATM/ATR and its target proteins.
EXAMPLE 10
[0308] Identification of Functional Relation Between Cancers and
AIM3
[0309] <10-1> Measurement of Expression Level of AIM3 in
Human Cancer Cell Lines
[0310] a) RT-PCR Analysis
[0311] To identify the functional relation between human cancers
and AIM3, the present inventors measured the level of AIM3 in
various cancer cell lines shown in Table 4, by RT-PCR. The RT-PCR
analysis was performed in the same method as in Example
<5-3>. TABLE-US-00004 TABLE 4 Derived p53 Cell line name from
function 1 HCT116 (human colon carcinoma cell line) Colon + 2 SW480
(human colon cancer cell lines) - 3 H23 (non-small cell lung cancer
cell line) Lungs - 4 H157 (non-small cell lung cancer cell line) -
5 A549 (human lung carcinoma cell line) + 6 H460 (human lung
carcinoma cell line) + 7 Raji (B-cell leukemia cell line) Lympho-
+/- 8 K-562 (human leukemia cell line) cytes -
[0312] As a result, as shown in FIG. 10a, the level of expression
of AIM3 was lower in HCT116, A549 and H460 cell lines.
Specifically, the level of AIM3 was low in the cells containing
active p53 (p53(+), i.e., HCT116, A549 and H460 cell lines), while
it was normal in the cells lacking active p53(p53(-), i.e., SW460,
H23, H157 and K-562 cell lines). Also in Raji cells containing
partially activated p53(Bhatia et al., FASEB J., 7:951-956, 1993),
the level of AIM3 was in the middle of that of p53(+) cells and
p53(-) cells. These results suggest that the expression level of
AIM3 has a functional relation with p53. Also, these results
further support that the aberration in either one of AIM3 or p53
may be sufficient to transform the cells, and AIM3 and p53 work in
the same signal transduction pathway.
[0313] Furthermore, the expression level of AIM3 was analyzed by
Western blot only in the lung cancer cell lines among the cell
lines in Table 4. And the results coincided with that of the above
RT-PCR analysis(data not shown).
[0314] These results suggest that the expression level of AIM3 in
some cancer cell lines is reduced.
[0315] b) Genomic PCR Analysis
[0316] To have a clue to the possible cause for the low expression
level of AIM3 in some cancer cell lines, the present inventors
compared the DNA content for the AIM3 gene by PCR. On H157, H460,
HCT116, A549 and DU145 cell lines, genomic DNA analysis was
performed in the same method as in Example <2-2>. As a
control group, an actin gene was used.
[0317] As a result, as shown in FIG. 10b, the H460 and A549 cell
lines contained less amount of AIM3 DNA than that of other cell
lines. This indicates that the two cell lines may have lost one
allele for AIM3.
[0318] <10-2> Measurement of Expression Levels of AIM3 and
p21 in Tissues Isolated From Cancer Patients
[0319] Thereafter, the expression levels of AIM3 and p21 in the
tissues isolated from cancer patients were examined. Total RNA was
extracted from the leukocytes of 9 leukemia patients (five
patients: acute promyelocytic leukemia (APML), and four patients:
chronic myelocytic leukemia (CML)). Then, RT-PCR was performed in
the same method as in Example <5-3>. In RT-PCR for p21,
primers shown in SEQ ID NO: 16 and SEQ ID NO: 17 were used.
[0320] As a result, as shown in FIG. 10c, the low level of AIM3 was
observed in the tissues of 3 patents. In this case, the expression
of p21, a target gene of p53, was also strongly suppressed. This
demonstrates again that AIM3 is functionally involved in the
regulation of p53.
[0321] <10-3> Comparative Measurement of Expression Levels of
AIM3 in Normal Tissue and Cancer Region From Liver Cancer
Patients
[0322] Since solid tumors were also found in AIM3.sup..+-. mice
although the frequency was much lower, the present inventors also
compared the expression levels of AIM3 in the cancer region with
that in normal tissue isolated from liver cancer patients by
RT-PCR. As a control group, the expression level of actin was also
measured. From the analysis of 25 different patient samples, a
cancer-specific reduction of AIM3 was observed in 12 samples. The
results for exemplary 8 samples are shown in FIG. 10d.
[0323] All these results in this Example suggest that a low level
of expression of AIM3 is associated with various human cancer cell
lines and patient tissues at high frequency. Also, these results
indicate that the measurement of the expression level of AIM3
allows for the diagnosis of cancers.
INDUSTRIAL APPLICABILITY
[0324] As described above, it was found in the present invention
that AIM3 acts as a powerful tumor suppressor. The AIM3 protein
binds to the FAT domain of ATM/ATR so as to activate ATM/ATR, thus
inducing the expression of p53, tumor suppressor protein.
Accordingly, the AIM3 protein or a nucleic acid encoding the
protein will be useful for cancer therapy. Furthermore, it will be
useful as targets for the development of anticancer drugs and as
diagnostic markers of various cancers.
Sequence CWU 1
1
17 1 174 PRT Homo sapiens 1 Met Ala Ala Ala Ala Glu Leu Ser Leu Leu
Glu Lys Ser Leu Gly Leu 1 5 10 15 Ser Lys Gly Asn Lys Tyr Ser Ala
Gln Gly Glu Arg Gln Ile Pro Val 20 25 30 Leu Gln Thr Asn Asn Gly
Pro Ser Leu Thr Gly Leu Thr Thr Ile Ala 35 40 45 Ala His Leu Val
Lys Gln Ala Asn Lys Glu Tyr Leu Leu Gly Ser Thr 50 55 60 Ala Glu
Glu Lys Ala Ile Val Gln Gln Trp Leu Glu Tyr Arg Val Thr 65 70 75 80
Gln Val Asp Gly His Ser Ser Lys Asn Asp Ile His Thr Leu Leu Lys 85
90 95 Asp Leu Asn Ser Tyr Leu Glu Asp Lys Val Tyr Leu Thr Gly Tyr
Asn 100 105 110 Phe Thr Leu Ala Asp Ile Leu Leu Tyr Tyr Gly Leu His
Arg Phe Ile 115 120 125 Val Asp Leu Thr Val Gln Glu Lys Glu Lys Tyr
Leu Asn Val Ser Arg 130 135 140 Trp Phe Cys His Ile Gln His Tyr Pro
Gly Ile Arg Gln His Leu Ser 145 150 155 160 Ser Val Val Phe Ile Lys
Asn Arg Leu Tyr Thr Asn Ser His 165 170 2 525 DNA Homo sapiens 2
atggcggcgg ccgcagagtt gtcgctactg gagaagtccc tgggactgag taaggggaat
60 aaatacagtg ctcagggcga gcgacagatt ccagttcttc agacaaacaa
tggtccaagt 120 ctaacaggat tgactactat agcagctcat ctagtcaagc
aagccaacaa agaatatttg 180 ctggggagta ctgcagaaga aaaagcaatc
gttcagcagt ggttagaata cagggtcact 240 caagtagatg ggcactccag
taaaaatgac atccacacac tgttgaagga tcttaattca 300 tatcttgaag
ataaagtcta ccttacaggg tataacttta cattagcaga tatactattg 360
tactatggac ttcatcgctt tatagttgac ctgacagttc aagaaaagga gaaatatctt
420 aatgtgtctc gctggttttg tcacattcag cattatccag gcatcaggca
acatctgtct 480 agtgttgtct tcatcaagaa cagactatat actaattccc actga
525 3 29 DNA Artificial Sequence p18F-1 primer for AIM3 3
gccggacttc ctgctcaatc aaggtccta 29 4 32 DNA Artificial Sequence
p18R-1 primer for AIM3 4 ctagcgggtg gataagtagt agtttcctca tg 32 5
27 DNA Artificial Sequence LTR primer for gene trap vector 5
cgttacttaa gctagcttgc cacctac 27 6 32 DNA Artificial Sequence
p18F-2 primer 6 catgaggaaa ctactactta tccacccgct ag 32 7 32 DNA
Artificial Sequence p18R-2 primer 7 ccttcagcag agtctgggtg
tcttctttac tg 32 8 22 DNA Artificial Sequence p18-AAAf primer for
AIM3 quantitation 8 atgggtccaa gtctaacagg at 22 9 22 DNA Artificial
Sequence p18-AAAr primer for AIM3 quantitation 9 tgtcaggtct
tctataaagc ga 22 10 20 DNA Artificial Sequence AIM3 antisense F1
primer for antisense AIM3 10 tctgccagct acggccggaa 20 11 24 DNA
Artificial Sequence AIM3 antisense R3 primer for antisense AIM3 11
ggcttgcttg actagatgag ctgc 24 12 21 DNA Artificial Sequence
FAT-sense primer for FAT 12 atggccaagg tagctcagtc t 21 13 21 DNA
Artificial Sequence FAT-antisense primer for FAT 13 tctgcttctt
ctggctacct c 21 14 21 DNA Artificial Sequence C-terminal-ATM sense
primer 14 attacgggtg ttgaaggtgt c 21 15 18 DNA Artificial Sequence
C-terminal-ATM antisense primer 15 ccaagctttt cctgggaa 18 16 22 DNA
Artificial Sequence p21-1 primer for p21 16 atgtcagaac cgggtgggga
tg 22 17 22 DNA Artificial Sequence p21-2 primer for p21 17
gggcttcctc ttggagaaga tc 22
* * * * *