Note: Descriptions are shown in the official language in which they were submitted.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 26
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 2
CONTAINING PAGES 1 TO 26
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-1-
A NOVEL COMPONENT OF THE WG/WNT SIGNALING PATHWAY
BACKGROUND OF THE INVENTION
Wnt genes of vertebraten and invertebraten encode a large family of secreted,
cystein rich
proteins that play key roles as intercellular signaling molecules in a wide
variety of biological
processes (for an extensive review see (Wodarz and Nusse 1998). The first Wnt
gene,
mouse Wnt-1, was discovered as a proto-oncogene activated by integration of
mouse
mammary tumor virus in mammary tumors (Nusse and Varmus 1982). Consequently,
the
involvement of the Wg/Wnt pathway in cancer has been largely studied. With the
identification of the Drosophila polarity gene wingless as a wnt-1 homologue
(Cabrera,
Alonso et al. 1987; Perrimon and Mahowald 1987; Rijsewijk, Schuermann et al.
1987), it
became clear that Wnt genes are important developmental regulators. Thus,
although at first
glance dissimilar, biological processes like embryogenesis and carcinogenesis
both rely on
cell communication via identical signaling pathways.
In a current model of the canonical Wnt pathway, the secreted Wnt protein
binds to Frizzle
cell surface receptors and activates the cytoplasmic protein Dishevelled
(Dsh). Dsh then
transmits the signal to a complex of several proteins, including the protein
kinase
Shaggy(Sgg)/GSK3, the scaffold protein Axin and O-Catenin, the vertebrate
homologue of
Armadillo. In this complex (3-Catenin is targeted for degradation after being
phosphorylated
by Sgg. After Wnt signaling and the resulting down-regulation of Sgg activity,
(3-Catenin (or
its Drosophila homologue Armadillo) escape from degradation and accumulate
into the
cytoplasm. Free cytoplasmic R-Catenin translocates to the nucleus by a still
obscure
mechanism, and modulates gene transcription through binding the Tcf/Lef family
of
transcription factors (Grosschedl R 1999). Mutations in 0-catenin, APC, and
Axin have been
found in several human c cancers, suggesting that constitutive activation of
canonical Wnt
pathway contributes to human carcinogenesis (Uthoff SM, Eichenberger MR,
McAuliffe TL,
Hamilton CJ and Galandiuk S. (2001). Mol. Carcinog., 31, 56-62.
Binding of Wnt ligands to their receptors also trigger activation of
noncanonical pathways,
referred as Wnt signaling pathways that signal independently of 'e-catenin,
which may signal
through calcium flux, c-Jun NH2-terminal kinase, and G proteins. These
pathways might get
activated in parallel to the canonical, bCatenin dependent pathway in tumors
characterized
by Wnt ligand upregulation (Huguet EL, McMahon JA, McMahon AP, Bicknell R and
Harris
AL. (1994). Cancer Res., 54, 2615-2621.; Dale TC, Weber-Hall SJ, Smith K,
Huguet EL,
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-2-
Jayatilake H, Gusterson BA, Shuttleworth G, O'Hare M and Harris AL. (1996).
Cancer Res.,
56, 4320-4323; Vider BZ, Zimber A, Chastre E, Prevot S, Gespach C, Estlein D,
Wolloch Y,
Tronick SR, Gazit A and Yaniv A. (1996). Oncogene, 12, 153-158; Smith K, Bui
TD, Poulsom
R, Kakiamanis L, Williams G and Harris AL. (1999). Br. J. Cancer, 81, 496-
502). For instance,
frequent upregulation of Wnt-2 has been reported in human colorectal cancer
and gastric
cancers (Katoh M. (2001). Int. J. Oncol., 19, 1003-1007.). Moreover, Holcombe
et al.
(Holcombe RF, Marsh JL, Waterman ML, Lin F, Milovanovic T and Truong T.
(2002). MoI.
Pathol., 55, 220-226) recently analysed the expression of specific Wnt genes
in human colon
cancer and malignant melanoma by in situ hybridization and their results
suggest that Wnt-2
overexpression may be involved in human carcinogenesis (Pham K, Milovanovic T,
Barr RJ,
Truong T and Holcombe RF. (2003). Mol. Pathol., 56, 280-285.).
In addition to its role in cancerogenesis, Wnt signalling also plays a role in
skeletogenesis,
bone formation and fracture repair (Hartmann (2000), Holmen (2005)). For
instance,
upregulation of Wnt-proteins have been shown to correlate with pathobiology of
rheumatoid
arthritis and osteoarthritis (Sen et al. (2000), Nakamura (2005), Holmen
(2005)).
Currently, there are no known therapeutic agents effectively inhibiting the
Wnt pathway,
either by directly inhibiting (3-Catenin transcriptional activation or by
inhibiting pathway
activation by Wnt ligands. This is partly due to the fact that many of the
essential
components required for its full activation and nuclear translocation are
still unknown.
Consequently, there is an urge to understand more about this pathway in order
to be able to
develop effective drugs against these highly malignant diseases.
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-3-
SUMMARY OF THE INVENTION
In order to identify new components required for Wg/Wnt signaling pathway
activation the
inventors used an approach for screening for recessive suppressors of the sev-
wg phenotype
(Drosophila melanogaster). In this approach a protein named 3L3 was found
which is
encoded by the gene CG6210. Said gene has homologues in all metazoans, and
more
interestingly, there is in all likelihood only one gene of this family in each
species. With
genetic tools it was confirmed that 3L3 plays a positive role in the Wg/Wnt
signaling
pathway, and in particular in the Wg/Wnt secretion pathway of all Wnt
proteins. Further it
has been shown that 3L3 physically interacts to Wnt-proteins. Therefore, 3L3
proteins are
very promising targets for developing drugs which up- or down-regulates Wnt
protein
secretion and thus inhibits both the canonical and non canonical Wnt pathway.
The invention relates to a method for screening for a substance that inhibits
or enhances the
secretion of a protein of the Wnt-family comprising the steps of:
a) bringing a candidate substance into contact with a nucleic acid molecule
with
the nucleotide sequence of SEQ ID. Nos 1, 2, 3 or 7 under conditions that
permit binding of said substance to said nucleic acid molecule; or
b) bringing a candidate substance into contact with nucleic acid molecule
coding
for a (poly)peptide with the amino acid sequences of SEQ ID. Nos 4, 5, 6 or 8
under conditions that permit binding of said substance to said nucleic acid
molecule; or
c) bringing a candidate substance into contact with a fragment of the nucleic
acid molecule according to a) or b) under conditions that permit binding of
said substance to said fragment, said fragment codes for the part of the
(poly)peptide with the amino acid sequences of SEQ ID. Nos 4, 5, 6 or 8
which effects Wnt-protein secretion; or
d) bringing a candidate substance into contact with a derivate of the nucleic
acid
molecule according to a) or b) or with a derivate of the fragment according to
c) under conditions that permit binding of said substance to said derivate; or
e) bringing a candidate substance into contact with a nucleic acid molecule,
with
a fragment or with a derivate which is at least 50 % homologous to the
nucleic acid molecule according to a) or b), to the fragment according to c)
or
to the derivate according to d), respectively;
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-4-
f) detecting if the candidate substance is having inhibitory activity or
enhancing
activity on the secretion of a Wnt-protein.
A derivate as stated under d) is a the nucleic acid molecule according to a)
or b) or a
fragment according to c) with an arbitrary molecule attached to it, said
candidate substance
showing an affinity to said derivate which is at the most 50% increased or
decreased
compared to the affinity between the candidate substance and the corresponding
unmodified
nucleic acid molecule or fragment, respectively.
"Percent (%) homologous" with respect to the following (poly)peptide sequences
is defined
as the percentage of amino acid residues in a candidate sequence that are
identical with the
amino acid residues in the sequences with SEQ ID. Nos 4, 5, 6 and 8 after
aligning the
sequence and introducing gaps, if necessary, to achieve the maximum percentage
sequence
identity, and not considering any conservative amino acid substitution as part
of the
sequence identity. The % identity values used herein can be generated by WU-
BLAST-2,
which was obtained from (Tatusova TA 1999). WU-BLAST-2 uses several search
parameters,
most of which are set to the default values.
In a similar manner, "percent (%) homologous" with respect to the mentioned
nucleic acid
sequences with the SEQ ID. Nos 1, 2, 3, and 7 is defined as the percentage of
nucleotide
residues in a candidate sequence that are identical with the nucleotide
residues in the said
nucleic acid sequences. The identity values used herein can be generated using
BLAST
module of WU-BLAST-2 set to the default parameters.
The (poly)peptides with the SEQ ID. Nos 4, 5, 6 and 8 encoded by nucleic acid
molecules
with the SEQ ID. Nos 1, 2, 3 and 7, respectively, are representatives of a
novel family of
proteins present in invertebrate, for example in Drosophila, leeches, slugs,
snails and
earthworms, and in vertebrate organisms, for example in mammals including
humans, apes,
monkeys, dogs, cats, rabbits, goats, pigs, hamsters, cows, horses, sheep, mice
and rats. The
(poly)peptides with the SEQ ID. Nos 4, 5, 6 and 8 are hereinafter referred to
as 3L3 or WLS
(wntless) proteins. These proteins play an essential role in the Wg/Wnt
signaling pathway
and thus in the formation and maintenance of spatial arrangements and
proliferation of
tissues during development, and in the formation and growth of many human
tumors. The
(poly)peptide with the SEQ ID. No 4 is the 3L3 protein of Drosophila (3L3-PA),
as well as the
(poly)peptide with the SEQ ID. No 8(3L3-PB). The (poly)peptides with the SEQ
ID. Nos 5
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-5-
and 6 are the 3L3 proteins of caenorhabditis (C.) elegans and human,
respectively, which
are structural and functional homologous of Drosophila 3L3.
In one embodiment of the invention, the nucleic acid molecule, the fragment or
the derivate
as stated under step e) is at least 52 % homologous to the nucleic acid
molecule according
to a) or b), to the fragment according to c) or to the derivate according to
d), respectively,
preferably at least 55 % homologous, more preferably at least 60 % homologous,
even more
preferably at least 65 % homologous, yet even more preferably at least 70 %
homologous.
In another preferred embodiment, the nucleic acid molecule, the fragment or
the derivate as
stated under step e) is at least 75 % homologous to the nucleic acid molecule
according to
a) or b), to the fragment according to c) or to the derivate according to d),
respectively,
preferably at least 80 % homologous, more preferably at least 85 % homologous,
even more
preferably at least 86 % homologous, yet even more preferably at least 87 %
homologous.
In a further preferred embodiment, the nucleic acid molecule, the fragment or
the derivate
as stated under step e) is at least 88 % homologous to the nucleic acid
molecule according
to a) or b), to the fragment according to c) or to the derivate according to
d), respectively,
preferably at least 89 % homologous, more preferably at least 90 % homologous,
even more
preferably at least 91 % homologous, yet even more preferably at least 92 %
homologous.
In a further preferred embodiment, the nucleic acid molecule, the fragment or
the derivate
as stated under step e) is at least 93 % homologous to the nucleic acid
molecule according
to a) or b), to the fragment according to c) or to the derivate according to
d), respectively,
preferably at least 94 % homologous, more preferably at least 95 % homologous,
even more
preferably at least 96 % homologous, yet even more preferably at least 97 %
homologous.
In a further preferred embodiment, the nucleic acid molecule, the fragment or
the derivate
as stated under step e) is at least 98 % homologous to the nucleic acid
molecule according
to a) or b), to the fragment according to c) or to the derivate according to
d), respectively,
preferably at least 99 % homologous.
The invention further relates to a method for screening for a substance that
inhibits or
enhances the secretion of a protein of the Wnt-family comprising the steps of:
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-6-
a) bringing a candidate substance into contact with a (poly)peptide with the
amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8 under conditions that permit
binding of said substance to said (poly)peptide; or
b) bringing a candidate substance into contact with a (poly)peptide fragment
of
the (poly)peptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8
under conditions that permit binding of said substance to said (poly)peptide
fragment, said fragment comprises the part of the (poly)peptide with the
amino acid sequences of SEQ ID. Nos 4, 5, 6 or 8 which effects Wnt-protein
secretion; or
c) bringing a candidate substance into contact with a derivate of the
(poly)peptide according to a) or with a derivate of the (poly)peptide fragment
according to b) under conditions that permit binding of said substance to said
derivate; or
d) bringing a candidate substance into contact with a (poly)peptide, with a
(poly)peptide fragment or with a derivate which is at least 50 % homologous
to the (poly)peptide according to a), to the (poly)peptide fragment according
to b) or to the derivate according to c), respectively; and
e) detecting if the candidate substance is having inhibitory activity or
enhancing
activity on the secretion of a Wnt-protein or if the candidate substance is
having inhibitory activity or enhancing activity on the binding between the
(poly)peptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8 and a
Wnt protein.
A derivate as stated under c) is a (poly)peptide with the amino acid sequence
of SEQ ID. Nos
4, 5, 6 or 8, or a (poly)peptide fragment according to b) with an arbitrary
molecule attached
to the N- or C-terminal part or to a side chain of an amino acid, and a
candidate substance
showing an affinity to said derivate which is at the most 50% increased or
decreased
compared to the affinity between the candidate substance and the corresponding
unmodified
(poly)peptide or (poly)peptide fragment, respectively.
In one embodiment of the invention, the (poly)peptide, the (poly)peptide
fragment or the
derivate as stated under step d) is at least 52 % homologous to the
(poly)peptide according
to a), to the (poly)peptide fragment according to b) or to the derivate
according to c),
respectively, preferably at least 55 % homologous, more preferably at least 60
%
homologous, even more preferably at least 65 % homologous, yet even more
preferably at
least 70 % homologous.
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-7-
In another preferred embodiment, the (poly)peptide, the (poly)peptide fragment
or the
derivate as stated under step d) is at least 75 % homologous to the
(poly)peptide according
to a), to the (poly)peptide fragment according to b) or to the derivate
according to c),
respectively, preferably at least 80 % homologous, more preferably at least 85
%
homologous, even more preferably at least 86 % homologous, yet even more
preferably at
least 87 % homologous.
In a further preferred embodiment, the (poly)peptide, the (poly)peptide
fragment or the
derivate as stated under step d) is at least 88 % homologous to the
(poly)peptide according
to a), to the (poly)peptide fragment according to b) or to the derivate
according to c),
respectively, preferably at least 89 % homologous, more preferably at least 90
%
homologous, even more preferably at least 91 % homologous, yet even more
preferably at
least 92 % homologous.
In a further preferred embodiment, the (poly)peptide, the (poly)peptide
fragment or the
derivate as stated under step d) is at least 93 % homologous to the
(poly)peptide according
to a), to the (poly)peptide fragment according to b) or to the derivate
according to c),
respectively, preferably at least 94 % homologous, more preferably at least 95
%
homologous, even more preferably at least 96 % homologous, yet even more
preferably at
least 97 % homologous.
In a further preferred embodiment, the (poly)peptide, the (poly)peptide
fragment or the
derivate as stated under step d) is at least 98 % homologous to the
(poly)peptide according
to a), to the (poly)peptide fragment according to b) or to the derivate
according to c),
respectively, preferably at least 99 % homologous.
Further, the invention relates to an antibody which specifically binds to the
(poly)peptide
according to step a), to the (poly)peptide fragment according step b), to the
derivate
according to step c) or to the (poly)peptide, the (poly)peptide fragment or
the derivate
according to step d), or to a (poly)peptide domain of the (poly)peptide with
the amino acid
sequence of SEQ ID. No 4, 5, 6 or 8, preferably to the (poly)peptide domain
which is
involved in Wnt-protein secretion or which binds to a Wnt-protein.
Aditionally, the invention relates to a siRNA with a target sequence being a
fragment of the
nucleic acid molecule coding for a (poly)peptide with the amino acid sequences
of SEQ ID.
Nos 4, 5, 6 or 8, preferably with a target sequence of SEQ ID Nos 9 or 10.
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-8-
In a further aspect, the invention relates to the use of said antibody or said
siRNA, the
nucleic acid molecule according to step a) or b) of the screening method, the
fragment
according to step c), the derivate according to step d) or the nucleic acid
molecule, the
fragment or the derivate according to step e), the (poly)peptide according to
step a) of said
screening method, the (poly)peptide fragment according to step b), the
derivate according
to step c) or the (poly)peptide, the (poly)peptide fragment or the derivate
according to step
d) as a drug.
The invention relates also to the use of said antibody or said siRNA, the
nucleic acid
molecule according to step a) or b) of the screening method, the fragment
according to step
c), the derivate according to step d) or the nucleic acid molecule, the
fragment or the
derivate according to step e), the (poly)peptide according to step a) of said
screening
method, the (poly)peptide fragment according to step b), the derivate
according to step c)
or the (poly)peptide, the (poly)peptide fragment or the derivate according to
step d) for the
preparation of a medicament for the treatment of Wnt-signaling related
disorders, preferably
cancer, bone or joint disorders or developmental disorders.
Preferred cancer types are Wnt-dependent cancer types, preferably colorectal
cancer, lung
cancer, nasopharyngeal carcinoma, preferably Wnt-2 dependent nasopharyngeal
carcinoma,
small intestinal adenocarcinoma, fundic gland polyps (gastric), gastric
carcinoma, gastric
(intestinal-like), gastric adenoma (without associated adenocarcinoma),
gastrointestinal
carcinoid tumor, esophageal adenocarcinoma, juvenile nasopharyngeal
angiofibromas,
melanoma, pilamatricomas, lung adenocarcinomas, ovarian carcinoma, uterine
cervix,
uterine endometrial, breast fibromatoses, prostate, thyroid carcinoma,
hepatoblastoma,
hepatocellular carcinoma, hepatocellular carcinoma associated with hepatitis
C,
medulloblastoma, desmoid tumor, Wilm's tumor (kidney), pancreatic (non-ductal
acinar cell
carcinomas), pancreatoblastoma and synovial sarcoma.
Preferred types of bone or joint disorders are osteoarthritis or rheumatoid
arthritis,
respectively.
Additionally, the invention relates to a pharmaceutical composition comprising
said antibody
or said siRNA.
In a further aspect, the invention relates to an assay for studying diseases
induced by an
altered Wnt secretion or for drug screening comprising the use of an organism,
for example
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-9-
a vertebrate or an invertebrate organism, preferably selected from the group
comprising
Drosophila, mice, rats, rabbits, chicken, frogs, pigs, sheep, worms, for
example C. elegans,
and fishes, for example zebrafish, or a cell line systems, preferably human
cell line systems,
said organisms or cell line system, respectively, showing increased or reduced
or no
expression of 3L3 or express a mutated 3L3 (poly)peptide in at least one
tissue or organ. "An
altered Wnt secretion" means for example an increased, decreased or disrupted
Wnt-protein
secretion.
In a preferred embodiment of the assay said organisms express the 3L3 gene
comprising at
least one of the nucleic acid molecules shown in SEQ. ID Nos 1 to 3 or 7 as a
transgene.
In another preferred embodiment of the assay said 3L3 gene comprises a
mutation selected
from the group consisting of deletions, point mutations, insertions and
inversions.
Still another aspect of the invention is a method for the modification of the
Wnt secretion of
a cell comprising the following step:
- bringing a cell into contact with a substance that inhibts or enhances Wnt-
protein secretion.
In a preferred embodiment of the method said substance is selected from said
antibody or
said siRNA, the nucleic acid molecule according to step a) or b), the fragment
according to
step c), the derivate according to step d) or the nucleic acid molecule, the
fragment or the
derivate according to step e), or the (poly)peptide according to step a), the
(poly)peptide
fragment according to step b), the derivate according to step c) or the
(poly)peptide, the
(poly)peptide fragment or the derivate according to step d).
The invention relates further to the use of the nucleic acid molecule
according to step a) or
b) of the screening method, the fragment according to step c), the derivate
according to
step d) or the nucleic acid molecule, the fragment or the derivate according
to step e), or
said antibody or said siRNA to regulate the Wnt-signaling pathway in cells,
preferably to
regulate cell fate determination or to control the development of stem cells
in vivo and in
vitro.
Additionally, the invention relates to the use of the (poly)peptide according
to step a) of said
screening method, the (poly)peptide fragment according to step b), the
derivate according
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
- 10-
to step c) or the (poly)peptide, the (poly)peptide fragment or the derivate
according to step
d) to regulate the Wnt-signaling pathway in cells, preferably to regulate cell
fate
determination or to control the development of stem cells in vivo and in
vitro.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. la-c: The CustalW protein alignment shows the high homology between the
3L3
protein of Drosophila, C. elegans and human. The putative signal sequence is
indicated by a dashed line above the sequence and the putative
transmembrane domains are underlined (TMHMM prediction).
Fig. 2: A) Normal adult pharates as control.
B) Adult pharates homozygous mutant for 3L3 showing that aristae of the
antennae are missing, and a rudimental development of all three leg pairs.
C) Adult pharates homozygous mutant for 3L3 showing that aristae of the
antennae are missing, a rudimental development of all three leg pairs, and a
wing-to-notum transformation (observed in 5-10% of cases) Embryo showing
the germline clone phenotype.
D) and E): wild-type situation showing a normal segmentation pattern.
F) and G) embryo mutant for 3L3 showing a classical Wg loss-of-function
phenotype with fusion of segments ("lawn of denticles" phenotype).
Fig. 3: A) Distalless-LacZ expression in a wild type disc.
B) Distalless-LacZ expression is reduced in 3L3 mutant tissue.
E) the same as B, but only the clone margin is shown.
D) Senseless protein expression in a wild type disc.
C) Senseless protein expression is suppressed in 3L3 mutant.
F) the same as B, but only the clone margin is shown.
Fig. 4: A) Wg protein expression in a wild type disc.
B) Wg protein expression is incresed in 3L3 mutant tissue, suggesting that in
these mutant cells Wg accumulates.
C) the same as B), but only the clone margin is shown.
D) extracellular Wg protein expression is not or only weakly incresed in 3L3
mutant tissue, suggesting that the accumulation of Wg seen in B-C is mainly
intracellular.
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-11-
E) the same as B, but only the clone margin is shown.
Fig. 5: TOP Flash Luciferase assay. Wnt3A-V5 and siRNA_h3L3 cotransfected
cells
show no induction of the Wnt signaling dependent Luciferase reporter gene as
the mock control, where empty vector (pcDNA3) was cotransfected with
siRNA_GFP. However, Wnt3A-V5 and siRNA_GFP cotransfected cells showed a
2-fold induction of the reporter gene compared to the mock control.
Fig. 6: Co-Immunoprecipitation, IP: a-HA, blot: a-V5. h3L3-HA is interacting
with
Wnt3A-V5 (Lane 1). Negative controls: no interaction could be observed
between CD2-HA and Wnt3A-V5 (Lane 2) and no band could be detected in
EGFP transfected cells (Lane 3).
Fig. 7: siRNA against the C. elegans homologue of 3L3. A) the gonad of a wt
worm.
siRNA against 3L3 leads to an abnormal arrangement of Oocytes within the
gonad (B) and sometimes to a defect in distal tip cell migration (C).
Fig. 8: TOP Flash Renilla Luciferase assay. WLS RNAi showed a clear down-
regulation
of Wg secretion compared to GFP RNAi.
Fig. 9: Secretion of Wnt3a is impaired upon knock-down of hWLS
A) The level of Wnt3a-V5 in the supernatant of HEK-293T cells is strongly
decreased by depletion of hWLS.
B) sihWLS abolishes secretion of Wntl-HA into the medium of COS-7 cells.
C) Application of sihWLS strongly decreases secretion of Wnt5a-HA compared
to siGFP.
Fig. 10: B-I) Cell surface stainings of HEK-293T cells. Wnt3a-V5 cannot reach
the cell
surface of cells co-transfected with sihWLS (B) whereas cotransfection of
siGFP leads to detectable levels of Wnt3a-V5 at the cell surface (C).
Wnt3aC77A-V5 is neither detectable at the surface of cells treated with
sihWLS (D) nor siGFP (E). (F-I) HA-CD2 is detected on the cell surface (F,G)
while CD2-HA is not (H,I); CD2-HA can be readily detected by conventional
staining (not shown). Surface levels of HA-CD2 are not affected by sihWLS
(F).
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-12-
Fig. 11: RNAi against the C. elegans WLS ortholog phenocopies the ABar spindle
orientation defect of mom-2 and mom-3 mutant embryos Embryos are shown
anterior to the left and ventral up. (A) Wild-type embryo, the ABar spindle
(yellow) is oriented perpendicular to the spindle of ABpr (blue), i.e.
perpendicular to the optical section shown (indicated by the yellow dot). (B)
In RNAi(mom-2) injected animals, the two spindles are in parallel orientation.
(C,D) In mom-3(or78) mutant embryos (C) as well as in RNAi(R06B9.6)
embryos (D) the ABar and ABpr spindles are also in parallel orientation.
Fig. 12: Secretion of Wnt3a is impaired upon knock-down of hWLS
A) Lysate and culture medium of HEK-293T cells cotransfected with Shh and
sihWLS show no alteration in production and secretion of Shh compared to
controls (siGFP).
B) Secretion of SSHA-dTNFAN (TNF-FLAG) is not affected by depletion of
WLS.
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-13-
DETAILED DESCRIPTION OF THE INVENTION
The Wnt signaling cascade is essential for the development of both vertebrates
and
invertebrates, and has been implicated in tumorigenesis. The Drosophila Wnt
genes are one
of the best characterized within the Wnt-protein family, which includes more
than hundred
genes. In the Drosophila embryo, Wg is required for formation of parasegment
boundaries
and for maintenance of engrailed (en) expression in adjacent cells. The
epidermis of embryo
defective in Wg function shows only a rudimentary segmentation, which is
reflected in an
abnormal cuticle pattern. While the ventral cuticle of wild type larvae
displays denticle belts
alternating with naked regions, the cuticle of Wg mutant larvae is completely
covered with
denticles. During imaginal disc development, Wg controls dorso-ventral
positional
information. In the leg disc, Wg patters the future leg by the induction of
ventral fate (Struhl
and Basler 1993). In animals with reduced Wg activity, the ventral half of the
leg develops
into a mirror image of the dorsal side (Baker 1988). Accordingly, reduced Wg
activity leads
to the transformation of wing to notal tissue, hence the name of the gene
(Sharma and
Chopra 1976). In the eye disc, Wg suppresses ommatidial differentiation in
favor of head
cuticle development, and is involved in establishing the dorso-ventral axis
across the eye
field (Heberlein, Borod et al. 1998).
Additional genes have been implicated in the secretion, reception or
interpretation of the Wg
signaling. For instance, genetic studies in Drosophila revealed the
involvement of frizzled
(Dfz), Dishevelled (dsh), shaggy/zeste-white-3 (sgg/zw-3), armadillo (arm),
adenomatous
polyposis coli (E-apc), axin, and pangolin (pan) in Wg signaling. The genetic
order of these
transducers has been established in which Wg acts through Dsh to inhibit Sgg,
thus relieving
the repression of Arm by Sgg, resulting in the cytoplasmic accumulation of Arm
and its
translocation to the nucleus. In the nucleus Arm interacts with Pan to
activate transcription
of target genes. Vertebrate homologues have been identified for all these
components (for
an updated review see (Peifer and Polakis 2000), suggesting that novel
identified members
of the Drosophila signaling pathway may likely have vertebrate counterparts.
Mutations leading to nuclear accumulation of the mammalian homologue of Arm, R-
Catenin,
and consequently to constitutive activation of the Wg/Wnt pathway have been
observed in
many types of cancer, including colon cancer, breast cancer, melanoma,
hepatocellular
carcinoma, ovarian cancer, endometrial cancer, medulloblastoma pilomatricomas,
and
prostate cancer (Morin 1999; Polakis, Hart et al. 1999). It is now apparent
that deregulation
of the Wnt signaling is an important event in the genesis of these
malignancies. However,
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-14-
there are still no known therapeutic agents effectively inhibiting the Wnt
pathway, either by
directly inhibiting R-Catenin transcriptional activation or by inhibiting
pathway activation by
Wnt ligands. This is partly due to the fact that many of the essential
components required
for its full activation are still unknown.
In order to identify new components required for Wingless activation the
inventors used a
Drosophila genetic approach to screen for recessive suppressors of the sev-wg
phenotype of
Drosophila melanogaster. In this approach a protein referred hereinafter to as
3L3 was
found which is encoded by the gene CG6210. Said gene has homologues in all
metazoans,
and more interestingly, there is in all likelihood only one gene of this
family in each species.
With genetic tools the inventors confirmed the role of 3L3 as a positiv
component of the
Wg/Wnt signaling pathway, and in particular its importance in the secretion of
the Wnt-
proteins. Further, it was shown by means of a binding study that 3L3 and Wnt
physically
interact.
In the screen for recessive suppressors of the sev-wg phenotype the inventors
found 3
different alleles of the 3L3 complementation group. Two alleles have mutations
in the so far
uncharacterized gene CG6210. CG6210 encodes for a protein which consists of a
signal
sequence at the N-terminus and 7 putative transmembrane domains. One allele of
the 3L3
complementation group, su20.53, contains a 47 bp deletion which leads to a
frameshift in
the last amino acid residue of the signal sequence and a premature stop codon.
The other
suppressor su20.54 has a point mutation that leads to an amino acid residue
exchange from
Prolin to Serin in the first transmembrane domain. Different bioinformatics
programs led to
the conclusion that this protein belongs to a family of proteins of unknown
function and
interestingly, there is always only one protein of this family per species.
Furthermore, the
protein is very well conserved in the different species, especially some of
the extracellular
sequences and the transmembrane-spanning domains (Fig. la-c), indicating that
its function
is highly specific and conserved.
Drosophila embryos are progressively subdivided into reiterated segments by
the localized
activities of several genes. Wingless (Wg) and Hedgehog (Hh) are the most
important genes
of the so-called segment polarity gene group, which eventually divides the A/P
axis into 14-
15 stripes prefiguring the future segments. As a consequence, the loss of Wg
or Hh function
leads to a disruption of the formation of the repeating exoskeletal structures
on the ventral
side of the embryo, which is the most obvious phenotypic outcome of the
activity of the
segment polarity genes.
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
- 15-
Genetic experiments called "germline clones" allow the inventors to generate
Drosophila
mutant embryos, which completely lack the protein of interest (in the present
case 3L3) and
then to study the segmentation pattern. In particular "germline clones" are
necessary to get
rid of the maternal component, which is often present in embryo for many
genes.
It was observed that these 3L3 mutant embryos show a fusion of the segments,
typical of a
Wg or Hh loss of function (Fig. 2 D to G). This results leads to the
conclusion that 3L3 is a
positive component of the Wg/Wnt or Hh signaling pathway.
Wg plays a pivotal role also in the development of adult appendages of
Drosophila, such as
antennae, legs, and wings. Therefore inventors studied the phenotype of 3L3
mutant adult
pharates, which represent flies just before hatching. They observed that all
the appendages,
whose development depends on Wg function, are abnormal (Fig. 2 A to C). In
particular
aristae of antennae are missing, legs are shorter and show segmentation
defects, and in 5%
of the cases a so-called "wing-to-notum" transformations was observed, which
are
pathognomonic for a Wg loss of function. These findings give evidence for the
conclusion
that 3L3 is a positive component in the Wg/Wnt signaling pathway.
Adult appendages, e.g. wings and legs, are formed in the larva by imaginal
cells, which are
organized in sack-like epithelia called "imaginal discs". In particular, the
wing imaginal disc
comprises around 20 cells when it is formed during embryonic development and
proliferates
during the three larval instars to generate a disc of ca. 75'000 cells at the
end of the larval
life. In these imaginal discs, Wingless behaves as a morphogen, whose spatial
concentration
varies and to which cells respond differently at different threshold
concentrations.
The inventors used in the experiments wing imaginal discs to study the effect
of 3L3 mutant
tissue on the expression of target genes, i.e. genes, which are activated by
Wg. Two kinds of
target genes are distinguished in the wing imaginal discs. On the one hand
there are "long-
range target genes" (such as Distalless), which are expressed also at lower Wg
concentrations, and therefore are expressed at several cell-diameter distance
from the Wg
source. On the other hand, there are target genes, which need a high level of
the
morphogen for their expression (so called "short-range target genes" (such as
Senseless).
After inducing 3L3 mutant clone formation in wing imaginal discs and the
expression of
Distalless-lacZ as a marker for the long range target genes and of the
Senseless as a marker
for the short range target genes (Fig. 3 A to F) was studied. In this
experiment a reduction
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-16-
of Distalless-acZ and Senseless expression was observed, but only if the
mutant tissue
involved also the the Wg producing cells. These results give evidence that 3L3
in all
likelihood play an important role in the production of the Wg morphogen.
To confirm the importance of 3L3 in Wg production, we tried to rescue flies
were selected
expressing 3L3 only in Wg producing cells. The rest of the animal was
homozygous mutant
for 3L3. The result was a complete rescue, what is a reference for the
conclusion that the
function of 3L3 is necessary only in Wg producing cells.
Further, the effect of 3L3 mutant tissue on the expression of Wg protein in
wing imaginal
discs was studied (Fig. 4 A to E). It was observed that in 3L3 mutant clones
there is an
increase in the amount of Wg expression. To better characterize this result,
the inventors
performed a similar experiment and looked at the wg-lacZ expression, which
reveals the
level of transcription of wg mRNA. The fact that no increase of the wg-lacZ
expression in 3L3
mutant tissue was present warrants the assumption that the increased staining
for Wg in
mutant tissue is due to a post-transcriptional process.
Finally, in order to distinguish whether the accumulation of Wg takes place
intra- or extra-
cellularly, the inventors examined Wg distribution using a different protocol
which allows
detection of extra-cellular proteins only however an accumulation of extra-
cellular Wg in 3L3
mutant clones was not detectable.
These results suggest a role for 3L3 in the secretion of Wg, since its loss of
function leads to
an accumulation of Wg inside the Wg producing cells.
To test whether the human homologue of 3L3 (h3L3) is involved in Wnt signaling
a Wnt
reporter gene assay (TOP-flash) was performed. This is a luciferase based
assay with 5 TCF
binding sites in front of the luciferase gene. The activity of the Wnt
signaling pathway can be
measured by the luminescence of the luciferase. From preceding discussed
results it was
known that 3L3 is important in Drosophilas Wnt producing cells. In order to
mimic Wnt
producing and receiving cells, one batch of human 293T cells was transfected
with mWnt3A-
V5 and siRNA against h3L3, another batch of cells was transfected with the TOP-
flash
reporter construct. 24 hours after transfection these two batches of cells
were mixed and
after a further incubation of 24 hours the luciferase activity was measured.
If siRNA against
3L3 was cotransfected with Wnt3A in the 'producing' cells then the level of
the luciferase
activity was as low as if empty vector (instead of Wnt3A) and siRNA against
GFP were
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-17-
transfected. On the other hand if Wnt3A and siRNA against GFP were transfected
a clear
induction of the reporter gene was observed (Fig. 5). These results indicate
that human (h)
3L3 is also a positive component of the Wnt pathway involved in the
production, not
reception, of the signal.
Since 3L3 is important in the Wnt producing cells it was investigated if h3L3-
HA interacts
with mouse (m) Wnt3A-V5. Co-immunoprecipitation experiments were performed and
the
results suggest that h3L3-HA interacts with mWnt3A-V5 whereas a negative
control with
CD2-HA, another membrane-spanning protein, did not interact with mWnt3A-V5
(Fig. 6).
In clones of wntless (WLS) mutant cells in Drosophila wing discs it was
observed an
accumulation of Wg in the Wg producing cells. In Kc cells we developed an
assay to measure
the secretion of Wg into the culture medium. The Renilla luciferase gene was
fused to the N-
terminus of wg and transfected into Kc cells. At the same time the cells were
treated with
dsRNA of WLS or GFP. WLS RNAi showed a clear down-regulation of Wg secretion
compared
to GFP RNAi (Fig. 8).
To determine whether this secretion defect was also the reason that in human
tissue culture
experiments depletion of WLS by RNAi resulted in the inability to activate the
Wnt signaling
cascade, we analysed the medium of Wnt secreting cells. HEK293T were
cotransfected with
siRNA and either Wnt3a-V5, Wntl-HA or Wnt5a-HA. The secretion of all these
Wnts was
significantly reduced when siRNA against WLS was cotransfected compared to
siRNA against
GFP. These results also indicate that in HEK293T cells the secretion of Wnts
is dramatically
reduced when WLS is depleted (Fig. 9; two different siRNAs (siRNAhWLS-A,
siRNAhWLS-B)
were validated by RT-PCR for their effectiveness to knock-down the expression
of the
endogenous hWLS gene in HEK-293T cells. Independent transfection of both
resulted in an
85% decrease of hWLS transcripts (not shown; siRNAhWLS-B was mostly used and
is
henceforth referred to as sihWLS, but siRNAhWLS-A showed the same effects).
Treatment of
responder cells with sihWLS, or treatment of producer cells with siRNA against
GFP (siGFP)
did not appreciably affect the outcome of the Wnt signalling assay.
To test whether in HEK293T cells WNT3a still can reach the extracellular
surface when WLS
is downregulated, we cotransfected siRNAs and Wnt3a-V5 and then stained only
the
extracellular fraction of WNT3a. As a control we used CD2 with either a HA-tag
at the N-
terminus (extracellular) or at the C-terminus (intracellular). With the
extracellular staining
protocol we used we could detect only the CD2 construct with the N-terminal HA-
tag. RNAi
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-18-
against WLS did not influence the secretion of CD2 to the extracellular
surface. In contrast,
the surface staining of of WNT3a-V5 was abolished in cells cotransfected with
siRNA against
WLS. Therefore we conclude, that WLS is necessary for the proper secretion of
WNT3a (and
probably other Wnt's as well) to the cell surface (Fig. 10).
In Drosophila wing discs dispatched clones showed a clear accumulation of Hh
ligand but, in
stark contrast, no accumulation of Hh could be observed in WLS clones. In
addition WLS
mutant flies have been rescued with a wg-Ga14::UAS-WLS construct. Expression
of WLS only
in wg-producing cells is sufficient to rescue the lethality and supports our
view that WLS is
specific for Wg signaling.
In human tissue culture only the secretion of Wnt's is affected but as shown
above the
secretion of CD2 is unaffected by WLS RNAi. Importantly, neither the secretion
of Shh nor
the secretion of TNF into the medium is influenced by WLS RNAi (Fig. 12).
These results
show that also the human orthologue of WLS is specific for Wnt signaling.
Our results also indicate that not only a small subset of Wnts is sensitive to
WLS loss of
function but that most if not all Wnt's are dependent on WLS. In C.elegans MOM-
3 has been
shown to be involved in many different, canonical and non-canonical Wnt
pathways such as
early blastomere polarization (Rocheleau, Thorpe), VPC specification
(Eisenmann and Kim),
Q neuroblast migration (Harris). These processes depend on three different
Wnt's: MOM-2
(Rochelau, Thorpe), LIN-44 (Jiang and Sternberg) and EGL-20 (Harris).
In C. elegans two Wnt signaling pathways can be distinguished, the canonical
pathway that
is postembryonically active in different developmental processes and the non-
canonical
pathway that can only be observed in embryonic development. Non-canonical Wnt
signaling
controls orientation of EMS division (the EMS cell is the precursor of the
future endoderm
and mesoderm precursor cells) and the fate of endoderm. Loss of non-canonical
Wnt
signaling leads to the loss of endoderm and the development of more mesoderm.
Several
components of this non-canonical Wnt signaling have been described. One of
them, mom-3
(more mesoderm-3), has been genetically characterized but has not been
molecularly
identified so far (Eisenmann and Kim 2000; Thorpe, Schlesinger et al. 1997).
By sequencing,
mutations in the homologue of 3L3 (c3L3) were found in different mom-3
alleles. Moreover,
RNAi against c3L3 led to typical loss of canonical and non-canonical Wnt
phenotypes such as
defects in distal tip cell migration and embryonic lethality. Together, these
results show that
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-19-
c3L3 is responsible for the mom-3 phenotype and that RNAi against c3L3 mimics
a loss of
mom-3 phenotype.
Wnt signaling in C.elegans is involved in many different developmental steps.
The earliest
step known is the MOM-2 dependent orientation of mitotic spindles and division
planes in the
four to eight cell embryo. Since mom-3 worms are mutant in the wntless
orthologue
R06B9.6, we injected dsRNA of R06B9.6 into the gonads of wt hermaphrodites. As
expected
all embryos analyzed injected with dsRNA of R06B9.6, or carrying a mutant
allele of mom-2
or mom-3 showed a parallel orientation of the Abar spindle to the ABpr
spindle. In contrast,
wt embryos had the two spindles oriented perpendicularly (Fig. 11).
In summary, the inventors found a polypeptide named 3L3 which is encoded by
the gene
CG6210. With genetic tools it was confirmed that 3L3 plays a role in the
Wg/Wnt signaling
pathway, and in particular in the Wg/Wnt secretion pathway of the Wnt
proteins. Further it
has been shown that 3L3 physically interacts to a Wnt-protein. Therefore 3L3
proteins are
very promising targets for developing drugs which positively or negatively
regulate the Wnt
pathway by regulating the secretion of the Wnt ligands.
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-20-
EXPERIMENTALPART
Example 1
Screen for recessive suppressors of the sev-wg phenotype:
The sev-wg transgene, which ectopically expresses Wg in the eye, leads to a
rough eye
phenotype that served as a phenotypic marker (Brunner et al., 1997) in the
screen. Males
carrying an FRT80 on the left arm and the sev-wg on the right arm of the 3rd
chromosome
were feeded for 12 hours with 21mM Ethyl methan sulfonate (EMS). 24 hours
after the
application of EMS the males were crossed to females encoding an eyeless-flp
recombinase
on the X chromosome and carrying an FRT80 M w+ on the 3d chromosome. Male
offspring
with the ey-flp and both FRT chromosomes were screened for a suppression of
the rough
eye phenotype. Complementation analysis between different alleles led to the
complementation group 3L3 consisting of two alleles.
Example 2
Homologues in all metazoans:
By Blast search (blastp) it was found that in every sequenced metazoan
organism there is
only one copy of this new protein family carrying the domain of unknown
function 1171
(DUF1171).
Example 3.
Membrane spanning domains:
TMHMM TOP predicted one N-terminal signal sequence and 7 putative
transmembrane
domains for CG6210-PB.
Example 4
Germline clones:
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-21-
It is an experimental procedure used to study the phenotype of embryo of
Drosophila
rnelanogasterin the complete absence of the protein of interest.
Female 3'd instar larvae carrying on one chromosome the FRT and the putative
mutation and
on the homologue chromosome the same FRT and the ovoD mutation were heat-
shocked for
1.5 hrs at 38 degrees. After hatching, virgins were crossed with males
carrying the same
puatative mutation and the FRT.
Female were allowed to lay eggs for one night on agar plate and embryos were
then
collected. They were then bleached for 3 minutes, and washed with tap water.
Then they
were put into water for 24 hrs. In order to remove the vitellum they were
shaken in a
biphasic solution containing equal amount of Methanol and Heptane. Embryos
were
subsequently washed 4x with Methanol and then 4x with Triton-X100 0.1%. After
6 hours
incubation at RT, they were finally mounted on a slide with Hoyer-lactate and
incubated for
12-24 hours at 60 C to digest protein: Cuticles were examined under the light
microscope.
Example 5
Wing imaginal discs to study the effect of 3L3 mutant tissue on the expression
of target
genes:
Imaginal discs are hollow sacs of cells that make adult structures during
metamorphosis.
They arise as pockets in the embryonic ectoderm and grow inside the body
cavity until the
larva becomes a pupa, at which point they turn inside out ("evaginate") to
form the body
wall and appendages.
Antibody staining of imaginal discs:
Larvae were dissected in Ringer solution on ice and fixed in 200 ul tubes with
PEM (200 pl)+
5% formaldehyde (10,7 p I) + 0.05% Triton X-100 (1 NI) for 20 min. After
washing 4x, plus
1 hour, plus 2-3x with PBT + Na-Azid, they were incubated over night with the
primary
antibody (diluted in PBT + Na-Azid) at 4 degrees. Larvae were washed 5x plus
30 min with
PBT + Na-Azid + 1% HINGS and incubated with the secondary antibody diluted in
PBT +
Na-Azid for 2 hours, at RT. Larvae were washed again 5x plus 2 hours with PBT
+ Na-Azid
and discs were mounted in PPDA. Staining in wing discs was analyzed on a
confocal
microscope.
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-22-
Example 6
Inducing 3L3 mutant clone formation in wing imaginal discs and studying the
expression of
Distalless-lacZ (wg-lacZ):
Mutant clones in the wing imaginal discs represent groups of cells, which are
mutant for the
protein of interest. In Drosophila melanogaster this is achieved by mitotic
recombination,
using the yeast-specific recombinase flippase (flp) driven by the heat-shock
promoter (hs-
tIp).
y w hsp-flp; allele 20.53 FRT80 / TM6b flies were crossed with y w hsp-flp;
nMyc [wfJ
Minute P[y-fJ FRT80/ TM6b, and females were allowed to lay eggs for two days.
Heat-shock at 37.0 degrees for 60 minutes was made 24-72 hours AEF, and then
larvae
were dissected 4 days later.
In the case of Distalless-lacZ the primary antibody P-galactosidase (1:2000
rabbit, polyclonal,
form Cappel ) was used.
In the case of wg-lacZ the antibody a-Wg (4D4) (1:1000 mouse, monoclonal,
DSHB) was
used.
Example 7
Confirmation of the importance of 3L3 in Wg production by means of the try to
rescue flies
expressing 30 only in Wg producing cells:
31_3 heterozygous mutant flies carrying the transgene wg-GAL4 (the
transcriptional activator
GAL4 is then expressed only in the cells, which express also Wg) were crossed
with flies,
which were also 3L3 heterozygous mutant and carried the trangene UAS-3L3 (UAS
binding
sites for the GAL4 protein activate the transcription of 3L3.
Example 8
Examination of Wg distribution:
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-23-
The protocol used to see extracellular Wg differs from the usual protocol in
the fact that the
staining with the primary antibody is performed in PBS at 4 degrees (in order
to block
endocytosis) for 30-60 minutes, and that fixation is done after the primary
antibody in PBS,
with 4% formaldehyde at 4 degrees for 20-30 minutes (Strigini and Cohen,
2000).
Example 9
The TOP Flash assay:
Cells were transfected by the Calcium-Phosphate method. 16 hours after
transfection cells
were washed and mixed. 24 hours after mixing, the cells were lysed with 1xPLB
from
Promega and analyzed.
Example 10
Co-immunoprecipitation:
Cells were transfected by the Calcium-Phosphate method. 36 hours after
transfection, cells
were lysed with RIPA buffer for 1 hour 30 minutes at 4 C. The lysate was
centrifuged for 30
minutes at 4 C and then incubated with Protein G Sepharose beads and rabbit a-
HA
overnight at 4 C. Beads were washed 4x with TBS and then Proteins were eluted
with SDS-
Loading buffer from the beads at 95 C for 10 minutes. Western blotting was
performed with
the mouse a-V5 antibody.
Constructs:
The mWnt3A cDNA sequence was cloned into pcDNA3 followed by a V5-tag and a His-
tag.
The h3L3 cDNA was cloned into pcDNA3 followed by a HA-tag. The siRNAs against
3L3 and
GFP were generated by Qiagen. CD2 was cloned with a C-terminal HA-tag into
pcDNA3.
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-24-
REFERENCES
Baker, N. E. (1988). 'Transcription of the segment-polarity gene wingless in
the imaginal
discs of Drosophila, and the phenotype of a pupal-lethal wg mutation."
Development 102(3):
489-97.
Cabrera, C. V., M. C. Alonso, et al. (1987). "Phenocopies induced with
antisense RNA identify
the wingless gene." Cell 50(4): 659-63.
Eisenmann, D. M., Kim, S. K. (2000). "Protruding vulva mutants identify novel
loci and Wnt
signaling factors that function during Caenorhabditis elegans vulva
development." Genetics
156(3): 1097-116.
Grosschedl R, E. Q. (1999). "Regulation of LEF-1TCF transcription factors by
Wnt and other
signals." Current Opinion in Cell Biology 11: 233-240.
Harris, J., Honigberg, L., Robinson, N., and Kenyon, C. (1996). Neuronal cell
migration in C.
elegans: regulation of Hox gene expression and cell position.
Development 122, 3117-3131.
Hartmann, C. and Tabin, C. J. (2000). õDual roles of Wnt signaling during
chondrogenesis in
the chicken limb." Development, 127(14), 3141-3159.
Heberlein, U., E. R. Borod, et al. (1998). "Dorsoventral patterning in the
Drosophila retina by
wingless." Development 125(4): 567-77.
Holmen, S. L. et al. (2005), õEssential role of beta-catenin in postnatal bone
acqusition." The
Journal of Biological Chemistry, 280 (22), 21162-21168.
Jiang, L. I., and Sternberg, P. W. (1999). Socket cells mediate spicule
morphogenesis in
Caenorhabditis elegans males. Dev Biol 211, 88-99.
Morin, P. J. (1999). "(3-catenin signaling and cancer." Bioessays 21(12): 1021-
30.
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-25-
Nakamura, Y., Nawata, M., Wakitani, S. (2005). "Expression profiles and
functional analyses
of Wnt-related genes in human joint disorders". American Journal of Pathology,
167(1), 97-
105.
Nusse, R. and H. E. Varmus (1982). "Many tumors induced by the mouse mammary
tumor
virus contain a provirus integrated in the same region of the host genome."
Cell 31(1): 99-
109.
Perrimon, N. and A. P. Mahowald (1987). "Multiple functions of segment
polarity genes in
Drosophila." Dev Biol 119(2): 587-600.
Peifer, M. and P. Polakis (2000). "Wnt signaling in oncogenesis and
embryogenesis--a look
outside the nucleus." Science 287(5458): 1606-9.
Polakis, P., M. Hart, et al. (1999). "Defects in the regulation of P-catenin
in colorectal
cancer." Adv Exp Med Biol 470: 23-32.
Potter, J. D. (1999). "Colorectal cancer: molecules and populations." Journal
of the National
Cancer Institute 91(11): 916-32.
Rijsewijk, F., M. Schuermann, et al. (1987). 'The Drosophila homolog of the
mouse
mammary oncogene int-1 is identical to the segment polarity gene wingless."
Cell 50(4):
649-57.
Rocheleau, C. E., Downs, W. D., Lin, R., Wittmann, C., Bei, Y., Cha, Y. H.,
Ali, M.,
Priess, J. R., and Mello, C. C. (1997). Wnt signaling and an APC-related gene
specify endoderm in early C. elegans embryos. Cell 90, 707-716.
Roose, J. and H. Clevers (1999). 'TCF transcription factors: molecular
switches in
carcinogenesis." Biochimica et Biophysica Acta 1424 (2-3): M23-37.
Sen, M., Lauterbach, K., El-Gabalawy, H., Firestein, G. S., Corr, M. Carson,
D. A. (2000).
,,Expression and function of wingless and frizzled homologs in rheumatoid
arthritis." PNAS,
97 (6), 2791-2796.
SUBSTITUTE SHEET (RULE 26)
CA 02600540 2007-09-11
WO 2006/097336 PCT/EP2006/002534
-26-
Sharma, R. P. and V. L. Chopra (1976). "Effect of the Wingless (wgl) mutation
on wing and
haltere development in Drosophila melanogaster." Dev Biol 48(2): 461-5.
Strigini, M., and. Cohen, S..M. (2000). Wingless gradient formation in the
Drosophila wing.
Curr. Biol. 10, 293-300.
Struhl, G. and K. Basler (1993). "Organizing activity of wingless protein in
Drosophila." Cell
72(4): 527-40.
Tatusova TA, M. T. (1999). "Blast 2 sequences--a new tool for comparing
protein and
nucleotide sequences." FEMS Microbiol Lett. 174: 247-250.
Thorpe, C.]., Schlesinger, A., et al (1997). "Wnt signaling polarizes an early
C. elegans
blastomere to distinguish endoderm from mesoderm." Cell;90(4): 695-705.
Thorpe, C. J., Schlesinger, A., and Bowerman, B. (2000). Wnt signalling in
Caenorhabditis elegans: regulating repressors and polarizing the cytoskeleton.
Trends Cell Biol 10, 10-17.
Waltzer, L. and M. Bienz (1999). "The control of (3-catenin and TCF during
embryonic
development and cancer." Cancer & Metastasis Reviews 18(2): 231-46.
Wodarz, A. and R. Nusse (1998). "Mechanisms of Wnt signaling in development."
Annual
Review of Cell & Developmental Biology 14: 59-88.
SUBSTITUTE SHEET (RULE 26)
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 26
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 2
CONTAINING PAGES 1 TO 26
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
