U.S. patent application number 11/522564 was filed with the patent office on 2007-04-05 for laminin chains: diagnostic uses.
This patent application is currently assigned to BioStratum, Inc.. Invention is credited to Pekka Kallunki, Charles Pyke, Karl Tryggvason.
Application Number | 20070077252 11/522564 |
Document ID | / |
Family ID | 27497119 |
Filed Date | 2007-04-05 |
United States Patent
Application |
20070077252 |
Kind Code |
A1 |
Tryggvason; Karl ; et
al. |
April 5, 2007 |
Laminin chains: diagnostic uses
Abstract
The instant invention provides for the identification,
diagnosis, monitoring, and treatment of invasive cells using the
laminin 5 gamma-2 chain protein or nucleic acid sequence, or
antibodies thereto.
Inventors: |
Tryggvason; Karl; (Oulu,
FI) ; Kallunki; Pekka; (Copenhagen, DK) ;
Pyke; Charles; (Hilleroo, DK) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE
32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
BioStratum, Inc.,
|
Family ID: |
27497119 |
Appl. No.: |
11/522564 |
Filed: |
September 18, 2006 |
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08317450 |
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Current U.S.
Class: |
424/155.1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C07K 2317/76 20130101; C12Q 2600/158 20130101; C12Q 2600/156
20130101; C12Q 2600/112 20130101; C07K 16/18 20130101; C07K 16/28
20130101; C12Q 1/6886 20130101 |
Class at
Publication: |
424/155.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method for intervention of .gamma.2 chain interactions of
invasive carcinogens with surrounding tissues by using anti-i2
chain antibodies to inhibit the .gamma.2 chain biological activity
of said invasive carcinogens.
2. The method of claim 1 wherein the antibody is monoclonal.
3. The method of claim 1 wherein the antibody is polyclonal.
4. A method for promoting adhesion of epithelial cells by exposing
said cells to intact laminin-5 molecules.
5. The method of claim 4 wherein said epithelial cells are squamous
carcinoma cells.
6. The method of claim 4 wherein the epithelial cells are human
keratinocytes.
Description
CROSS REFERENCE
[0001] This application claims the benefit of the filing date of
U.S. Provisional Application No. 60/175,005, filed Jan. 7, 2000.
This application is also a continuation-in-part of U.S. Application
Ser. No. 09/663,147, filed on Sept. 15, 2000, which is a
continuation of U.S. Application Ser. No. 08/800,593 filed Feb. 18,
1997, now U.S. Pat. No. 6,143,505, which is a divisional of U.S.
Application Ser. No. 08/317,450 filed Oct. 4, 1994, now U.S. Pat.
No. 5,660,982.
BACKGROUND OF THE INVENTION
[0002] Laminins are a family of basement membrane proteins which
function in cell differentiation, adhesion, and migration, in
addition to being true structural components (Tryggvason K., Curr.
Opn. Cell Biol., 1993, 5:877-882, this and all following references
are hereby incorporated by reference). The laminin molecule is a
cross-shaped heterotrimer consisting of one heavy chain
(.apprxeq.400 kd) and two light chains, .beta. and .gamma. (130-200
kd) (nomenclature according to Burgeson et al., Matrix Biol., 1994,
14:209-211). Laminins exist as several isoforms each having a
unique combination of .alpha., .beta. and .gamma. chains. Thus far,
ten genetically distinct laminin chains, .alpha.1-.alpha.5,
.beta.1-.beta.3 and .gamma.1-.gamma.2 are known.
[0003] In the laminin molecule the three chains are associated
through a carboxyl terminal coiled coil (long arm), most of the
chains having a free amino terminal short arm. Additionally, all
the .alpha. chains have a large globular G domain at the carboxyl
terminus. Laminin can contribute to the structural framework of the
basement membrane, but it is also believed to have a role in cell
differentiation, proliferation, adhesion and migration (Timpl, R.
& Brown, J. C. (1994) Matrix Biol. 14: 275-81, Yurchenco, P. D.
& O'Rear, J. J. (1994) Curr. Opin. Cell. Biol. 6: 674-81). Many
of the laminin chains have tissue- and cell-specific distribution
which may vary between different developmental stages, indicating
specific functions for the various chains and isoforms. Evidence
for tissue-specific roles of some of the laminin chains has come
from identification of mutations in the .alpha.2 chain gene in
muscular dystrophies in mouse and man (Xu, H., Wu, X. R., Wewer, U.
M. & Engvall, E. (1994) Nature Genet. 8: 297-301;
Heibling-Leclerc, A., Zhang, X., Topaloglu, H., Cruaud, C., Tesson,
F., Weissenbach, J., Tome', F., Schwartz, K., Fardeau, M.,
Tryggvason, K. & Guicheney, P. (1995) Nature Genet. 11:
216-218; Nissinen, M., Heibling-Leclerc, A., Zhang, X.,
Evangelista, T., Topaloglu H., Cruaud, C., Weissenbach, J.,
Fardeau, M., Tome', F. M. S., Schwartz, K., Tryggvason, K. &
Guicheney, P. (1996) Am. J. Hum. Genet. 58: 1177-1184), as well as
in the genes for the .alpha.3, .beta.3 and .gamma.2 chains in
epidermolysis bullosa (Pulkkinen, L., Christiano, A. M., Airenne,
T., Haakana, H., Tryggvason, K. & Uitto, J. (1994a) Nature
Genet. 6: 293-297; Pulkkinen, L., Christiano, A. M., Gerecke, D.,
Wagman, D. W., Burgeson, R. E., Pittelkow, M. R. & Uitto, J.
(1994b) Genomics 24: 357-60; Aberdam, D., Galliano, M. F., Vailly,
J., Pulkkinen, L., Bonifas, J., Christiano, A.M., Tryggvason, K.,
Uitto, J., Epstein, E. J., Ortonne, J. P. & Meneguzzi, G.
(1994) Nature Genet. 6: 299-304; Kivirikko, S., McGrath, J. A.,
Baudoin, C., Aberdam, D., Ciatti, S., Dunnill, M. G. S., McMillan,
J. R., Eady, R. A. J., Ortonne, J.-P., Meneguzzi, G., Uitto, J.
& Christiano, A. M. (1995) Hum. Mol. Genet. 4: 959-962; Vidal,
F., Baudoin, C., Miquel, C., Galliano, M.-F., Christiano, A. M.,
Uitto, J., Ortonne, J.-P. & Meneguzzi, G. (1995) Genomics 30:
273-280).
[0004] Laminin-5, is a unique subepithelial basement membrane
isoform with the molecular formula .alpha.3:.beta.3:.gamma.2 chains
(Burgeson, R. E., Chiquet, M., Deutzmann, R., Ekblom, P., Engel,
J., Kleinman, H., Martin, G. R., Meneguzzi, G., Paulsson, M.,
Sanes, J., Timpl, R., Tryggvason, K., Yamada, Y., & Yurchenco,
P. D. (1994) Matrix Biol. 14:209-211). Determination of the primary
structure of the human .alpha.3, .beta.3 and .gamma.2 chains has
revealed that all these chains are truncated in the short arm
relative to the corresponding chains of laminin-1 (Kallunki, P.,
Sainio, K., Eddy, R., Byers, M., Kallunki, T., Sariola, H., Beck,
K., Hirvonen, H., Shows, T. B. & Tryggvason, K. (1992) J. Cell
Biol. 119:679-693; Ryan, M. C., Tizard, R., VanDevanter, D. R.
& Carter, W. G. (1994) J. Biol. Chem. 269: 22779-22787;
Gerecke, D. R., Wagman, D. W., Champliaud, M. F. & Burgeson, R.
E. (1994) J. Biol. Chem). Additionally, the .gamma.2 chain exists
in two forms differing in the length of their carboxyl terminal end
due to alternative splicing Kallunki, P., Sainio, K., Eddy, R.,
Byers, M., Kallunki, T., Sariola, H., Beck, K., Hirvonen, H.,
Shows, T. B. & Tryggvason, K. (1992) J. Cell Biol. 119:
679-693; Ryan, M. C., Tizard, R., VanDevanter, D. R. & Carter,
W. G. (1994) J. Biol. Chem. 269: 22779-22787; Gerecke, D. R.,
Wagman, D. W., Champliaud, M. F. & Burgeson, R. E. (1994) J.
Biol. Chem; Airenne, T., Haakana, H., Sainio, K., Kallunki, T.,
Kallunki, P., Sariola, H. & Tryggvason, K. (1996) Genomics 32:
54-64). Immunolocalization of the laminin-5 protein (previously
termed kalinin, nicein or epiligrin) to anchoring filaments
(Verrando, P., Hsi, B., Yeh, C., Pisani, A., Serieys, N., &
Ortonne, J. (1987) Exp. Cell Res. 170:116-128; Carter, W. G., Ryan,
M. C. & Gahr, P. J. (1991) Cell 65: 599-610; Rousselle, P.,
Lunstrum, G. P., Keene, D. R. & Burgeson, R. E. (1991) J. Cell
Biol. 114:567-576) as well as epithelium-specific expression of the
.gamma.2 chain (Kallunki, P., Sainio, K., Eddy, R., Byers, M.,
Kallunki, T., Sariola, H., Beck, K., Hirvonen, H., Shows, T. B.
& Tryggvason, K. (1992) J. Cell Biol. 119: 679-693) already
implied its role as an epithelial attachment component. The
adhesion properties of laminin-5 have been demonstrated in several
cell attachment studies (Carter, W. G., Ryan, M. C. & Gahr, P.
J. (1991) Cell 65: 599-610; Rousselle, P., Lunstrum, G. P., Keene,
D. R. & Burgeson, R. E. (1991) J. Cell Biol. 114: 567-576;
Sonnenberg, A., Calafat, J., Janssen, H., Daams, H., van der
Raaij-Helmer, L. M. H., Falcioni, R., Kennel, S. J., Aplin, J. D.,
Baker, J., Loizidou, M. & Garrod, D. (1991) J. Cell Biol. 113:
907-917; Niessen, C. M., Hogervorst, F., Jaspars, L. H., De Melker,
A. A., Delwel, G. O., Hulsman, E. H., Kuikman, I. & Sonnenberg,
A. (1994) Exp. Cell. Res. 211: 360-367; Rousselle, P. &
Aumailley, M. (1994) J. Cell Biol. 125:205-214). The adhesive
function of laminin-5 has been shown to be mediated through
.alpha.3.beta.1 and .alpha.6.beta.4 integrins (Carter, W. G., Ryan,
M. C. & Gahr, P. J. (1991) Cell 65: 599-610; Sonnenberg, A.,
Calafat, J., Janssen, H., Daams, H., van der Raaij-Helmer, L. M.
H., Falcioni, R., Kennel, S. J., Aplin, J. D., Baker, J., Loizidou,
M. & Garrod, D. (1991) J. Cell Biol. 113: 907-917; Rousselle,
P. & Aumailley, M. (1994) J. Cell Biol. 125:205-214). Direct
evidence for the crucial role of laminin-5 for epithelial cell
attachment has come from the identification of mutations in the
genes of all the subunit chains (Pulkinen, L., Christiano, A.M.,
Airenne, T., Haakana, H., Tryggvason, K. & Uitto, J. (1994)
Nature Genet. 6: 293-297; Pulkkinen, L., Christiano, A. M.,
Gerecke, D., Wagman, D. W., Burgeson, R. E., Pittelkow, M. R. &
Uitto, J. (1994b) Genomics 24: 357-60; Aberdam, D., Galliano, M.
F., Vailly, J., Pulkkinen, L., Bonifas, J., Christiano, A. M.,
Tryggvason, K., Uitto, J., Epstein, E. J., Ortonne, J. P. &
Meneguzzi, G. (1994) Nature Genet. 6: 299-304; Kivirikko, S.,
McGrath, J. A., Baudoin, C., Aberdam, D., Ciatti, S., Dunnill, M.
G. S., McMillan, J. R., Eady, R. A. J., Ortonne, J.-P., Meneguzzi,
G., Uitto, J. & Christiano, A. M. (1995) Hum. Mol. Genet. 4:
959-962; Vidal F., Baudoin, C., Miquel, C., Galliano, M.-F.,
Christiano, A. M., Uitto, J., Ortonne, J.-P. & Meneguzzi, G.
(1995) Genomics 30: 273-280) in the Herlitz's variant of junctional
epidermolysis bullosa, a lethal skin blistering disease caused by
disruption of the epidermal-dermal junction. One and possibly the
only cell adhesion site of laminin-5 has been localized to the long
arm (Rousselle, P. & Aumailley, M. (1994) J. Cell Biol.
125:205-214; Rousselle, P., Golbik, R., van der Rest, M. &
Aumailley, M. (1995) J. Biol. Chem. 270:13766-13770). However, a
mutation in one junctional epidermolysis bullosa patient causing an
in-frame deletion of 73 residues from domains III and IV of the
short arm of the laminin .gamma.2 chain indicates a role for this
part of the chain for the anchorage of epithelial cells to the
extracellular matrix (Pulkkinen, L., Christiano, A. M., Airenne,
T., Haakana, H., Tryggvason, K. & Uitto, J. (1994) Nature
Genet. 6: 293-297).
[0005] By in situ hybridization the .gamma.-2 chain was found to be
expressed in epithelial cells of many embryonic tissues such as
those of skin, lung, and kidney (Kallunki et al., 1992, supra.),
and antibodies to kalinin/laminin 5, react with basement membranes
of the same tissues (Rousselle et al., 1991, supra.; Verrando et
al., Lab. Invest., 1991, 64:85-92).
[0006] The different laminin chains have been shown to have quite
varying tissue distribution as determined by immunohistological
studies, Northern, and in situ hybridization analyses. For example,
the A and M chains on the one hand, and the B1 (.beta.-1) and S
(.beta.-2) chains on the other, have been shown to be mutually
exclusive (see for example Vuolteenaho et al., J. Cell Biol., 1994,
124:381-394). In vitro studies have indicated that laminin mediates
a variety of biological functions such as stimulation of cell
proliferation, cell adhesion, differentiation, and neurite
outgrowth. The cellular activities are thought to be mediated by
cell membrane receptors, many of which are members of the integrin
family (Ruoslahti, E. J. Clin. Invest., 1991, 87:1-5; Mecham, R. P.
FASEB J., 1991, 5:2538-2546; Hynes, R. Cell, 1992, 69:11-25).
Recently a new nomenclature for describing laminins has been agreed
to as in the following Table 1 (after Burgeson et al., 1994,
supra): TABLE-US-00001 TABLE 1 Laminin Chains and Genes New
Previous Gene .alpha.1 A, Ae LAMA1 .alpha.2 M, Am LAMA2 .alpha.3
200 kDa LAMA3 .beta.1 B1 B1e LAMB1 .beta.2 S, B1s LAMB2 .beta.3 140
kDa LAMB3 .gamma.1 B2, B2e LAMC1 .gamma.2 B2t LAMC2
[0007] TABLE-US-00002 Heterotrimers of Laminin New Chains Previous
Laminin-1 .alpha.1.beta.1.gamma.1 EHS laminin Laminin-2
.alpha.2.beta.1.gamma.1 merosin Laminin-3 .alpha.1.beta.2.gamma.1
s-laminin Laminin-4 .alpha.2.beta.2.gamma.1 s-merosin Laminin-5
.alpha.3.beta.3.gamma.2 kalinin/nicein Laminin-6
.alpha.3.beta.1.gamma.1 k-laminin Laminin-7 .alpha.3.beta.2.gamma.1
ks-laminin
[0008] Cell migration is one of the biological functions proposed
for laminin (Timpl, R. & Brown, J. C. (1994) Matrix Biol. 14:
275-81). Cellular movement is required for various physiological
and pathological processes, such as during embryogenesis, wound
healing, angiogenesis and tumor invasion. Immunohistochemical and
in situ hybridization studies have shown induction of laminin-5
expression in migrating keratinocytes during wound healing (Ryan,
M. C., Tizard, R., VanDevanter, D. R. & Carter, W. G. (1994) J.
Biol. Chem. 269: 22779-22787; Larjava, H., Salo, T., Haapasalmi K.,
Kramer, R. H. & Heino J. (1993) Clin. Invest. 92: 1425-1435;
Pyke, C., Romer, J., Kallunki, P., Lund, L. R., Ralfkiaer, E.,
Dano, K. & Tryggvason, K. (1994) Am. J. Pathol. 145: 782-791).
The .gamma.2 chain of laminin-5 has also been shown to be strongly
expressed in malignant cells located at the invasion front of
several human carcinomas, as determined by in situ hybridization
and immunohistochemical staining Pyke, C., Romer, J., Kallunki, P.,
Lund, L. R., Ralfkiaer, E., Dano, K. & Tryggvason, K. (1994)
Am. J. Pathol. 145: 782-791; Pyke, C., Salo, S., Ralfkiaer, E.,
Romer, J., Dano, K. & Tryggvason, K. (1995) Cancer Res. 55:
4132-4139). Since laminin-1 has been found to inhibit keratinocyte
migration in vitro (Woodley, D. T., Bachmann, P. M. & O'Keefe,
E. J. (1988) J. Cell. Physiol. 136: 140-146), and as the laminin
.alpha.1, .beta.1 and .gamma.1 chains are only weakly expressed
throughout cancerous areas with no apparent correlation to sites of
invasion, laminin-5 has been proposed to have a role in the
migration event (Pyke, C., Romer, J., Kallunki, P., Lund, L. R.,
Ralfkiaer, E., Dano, K. & Tryggvason, K. (1994) Am. J. Pathol.
145: 782-791).
SUMMARY OF THE INVENTION
[0009] The instant invention provides; for methods of detecting
kalinin/laminin 5 expression in tissue comprising detecting a
signal from assayed tissue, such signal resulting from specifically
hybridizing tissue with an effective amount of a nucleic acid
probe, which probe contains a sense or antisense portion of
kalinin/laminin 5 gamma-2 nucleic acid sequence (Kallunki et al.,
1992, supra.). In particular, where the nucleic acid probe is DNA,
RNA, radio-labeled, enzyme labeled, chemiluminescent labeled,
avidin or biotin labeled, derived from human kalinin/laminin 5
gamma-2 nucleic acid sequence, incorporated into an
extrachromasomal self-replicating vector, a viral vector, is
linear, circularized, or contains modified nucleotides. In the
preferred embodiment the probes are linearized specific regions of
the .gamma.-2 gene.
[0010] The instant invention also provides for methods for
detecting the presence of invasive cells in tissue comprising
detecting a signal from assayed tissue, such signal resulting from
contacting tissue with an effective amount of a nucleic acid probe,
which probe contains a sense or antisense portion of
kalinin/laminin 5 .gamma.-2 nucleic acid sequence (Kallunki et al.,
1992, supra.). In particular, where the nucleic acid probe is DNA,
RNA, radio-labeled, enzyme labeled, chemiluminescent labeled,
avidin or biotin labeled, derived from human kalinin/laminin 5
gamma-2 nucleic acid sequence, incorporated into an
extrachromasomal self-replicating vector, a viral vector, is
linear, circularized, or contains modified nucleotides. In the
preferred embodiment the probes are linearized specific regions of
the .gamma.-2 gene. The instant method also provides for the
diagnosis of the absence of .gamma.-2 chain expression, useful for
the monitoring of therapies, and the progress of malignant cell
transformation leading to accurate determination of the extent of
invasive cell activity.
[0011] The instant invention further provides for a method for
detecting kalinin/laminin 5 expression in tissue comprising
detecting a signal from assayed tissue, such signal resulting from
contacting tissue with an effective amount of a labeled probe,
which probe contains an antibody immunoreactive with a portion of
linin/laminin 5 gamma-2 protein.
[0012] Further provided is a method for detecting invasive cells in
tissue comprising detecting a signal from assayed tissue, such
signal resulting from contacting tissue with an effective amount of
a labeled probe, which probe contains an antibody immunoreactive
with a portion of kalinin/laminin 5 gamina-2 protein. Also provided
is a method for detecting kalinin/laminin 5 in tissue comprising
detecting a signal from assayed tissue, such signal resulting from
contacting tissue with an effective amount of a labeled probe,
which probe contains an antibody immunoreactive with a potion of
kalinin/laminin 5 gamma-2 protein. Thus the method of the instant
invention provides for the absence of such signal as diagnostic for
the absence of invasive cells.
[0013] Further, the present invention provides for a method of
using the laminin-5 molecule to promote adhesion of cultured
epithelial and carcinoma cells.
[0014] Additionally, the present invention is directed to a method
for blocking migration of cells using antibodies against the
.gamma.-2 chain of the laminin-5 molecule. Evidence for the
relationship of .gamma.2 chain expression with cell migration was
obtained by the identification of an enhancer element in the LAMC2
gene in studies on promoter-reporter gene constructs in transgenic
mice.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A-D shows in situ hybridization of a specimen of
colon adenocarcinoma for .gamma.-2 chain mRNA using a S-35 labeled
anti-sense RNA probe derived from plasmid pbb2r-02.
Magnification:1A.times.100; 1B-1D.times.640.
[0016] FIGS. 2A-D shows in situ hybridization for .gamma.-2 chain
mRNA on sections of ductal mammary carcinoma (2A), malignant
melanoma (2B), squamous cell carcinoma of the skin (2C-2D), and
squamous cell carcinoma of the vulva (2E-2G). Magnification:
2C.times.100, all others .times.640. Photos marked by plain letter
ie. X, show in situ hybridization results for .gamma.-2 chain mRNA
on stained sections. Photos marked by the apostrophe letter, ie.
X', are the dark field images of the respective
photomicographs.
[0017] FIGS. 3A,A' is incisionally wounded mouse skin (72 hours
after wounding) showing signal for .gamma.-2 chain in keratinocytes
at the leading edge of the migrating epithelium (curved arrow).
Magnification: .times.640. FIG. 3A is a photo of in situ
hybridization on a stained section showing .gamma.-2 chain signal.
FIG. 3A' is a photo showing the dark field image of 3A.
[0018] FIGS. 4A-B shows the nucleic acid sequence for the .gamma.-2
chain cDNA and the derived amino acid sequence. FIG. 4A is the full
cDNA for the 5,200 base pair sequence, availible from
EMB/GenBank/DDBJ under the accession number Z15008. FIG. 4B is the
nucleotide and derived amino acid sequence of the alternative 3'
end sequence from cDNA crones providing a sequence of 4,316 base
pairs, availible from EMB/GenBank/DDBJ under the accession number
Z15009. Kallunki et al.,1992, supra) SEQ ID NOs:12,13,14&
15.
[0019] FIG. 5. Characterization of laminin-5 and recombinant
laminin .gamma.2 chain prepared and used in this study. HaCat cell
culture medium proteins and the recombinant laminin .gamma.2 chain
extracted from baculovirus-infected insect cells were purified as
described in Materials and Methods and studied on 6% polyacrylamide
gels. (A) Silver stained laminin-5 (LAM5) from HaCat cells was
resolved into two major bands of 165 kDa and 140-155 kDa.
Additionally, a weak band of 105 kDa could be observed. These bands
correspond in size to those of the .alpha.3 (165 kDa),
.beta.2+.beta.3 (155 kDa+140 kDa) and processed .gamma.2 (105 kDa)
chains of laminin-5. The recombinant .gamma.2 chain (rec.gamma.2)
produced by the baculovirus system showed a major silver stain band
of about 155 kDa).
[0020] FIG. 6. Efficiency of human laminin-5 and recombinant human
laminin .gamma.2 chain for attachment of HaCat keratinocytes and
KLN-205 squamous carcinoma cells in vitro. The attachment
efficiency was compared with the efficiency with which the cells
bound to laminin-1. Substrate concentrations (10 .mu.g/ml)
providing maximum attachment to laminin-1 and laminin-5 were used.
The results are presented as means +/- SD calculated from at least
four duplicate series, the values for laminin-1 were given the
arbitrary value of 100%.
[0021] FIGS. 7A-B. Effects of polyclonal .gamma.2 chain antibodies
on the migration of KLN-205 squamous carcinoma cells in Boyden and
Transwell chamber assays of migration. (7A) The two compartments of
the chemotactic Boyden chambers were separated by a type IV
collagen coated porous Nucleopore filter (pore size 8 .mu.m). The
cells (1.times.105) in MEM containing 0.1% BSA were placed in the
upper compartment, and laminin-1 (+/-) or fibronectin (-/+) in MEM
containing 0.1% BSA were added as chemoattractants to the lower
compartment. IgG against .gamma.2 chain domains III, I/II or
preimmune IgG was added to the upper compartment with the cells at
a concentration of 20 .mu.g/ml. After 8-hour incubation at
.sub.37.degree. C. the filters were removed and migration of cells
to the lower surface of the filter were quantitated. The data are
expressed as percentage of migrated cells (+/- SD (bars)) per high
power field, setting migration in the presence of pre-immune IgG as
100%. Cells were counted in ten randomly selected high power fields
to triplicate assays. (7B), Effects of exogenous laminin-5 on cell
migration in a Transwell assay. The lower side of the membrane was
coated with EHS type IV collagen, and the lower compartment was
filled with 2.5 .mu.g/ml laminin-5 as a chemoattractant. Pre-immune
IgG, IgG against the .gamma.2 chain domains III or I/II were added
to the upper chamber containing the cells. Following 16 hour
incubation the cells were fixed and cells at the lower side of the
membrane were counted (12 fields +/- SD).
[0022] FIG. 8. LAMC2 Promoter-reporter gene constructs used for
studies of expression in transgenic mice. Top, nucleotide sequence
of the 5' region of the LAMC2 gene and its immediate upstream
region (SEQ. ID. NO.:20). The bent arrow indicates the site of
transcription initiation. The GATAA motif is boxed and the AP-1,
Sp1 and CTF motifs are underlined The translation initiator codon
ATG in exon 1 for methionine is indicted by a double underline. The
first amino acids of the protein are shown with the single letter
code beneath the corresponding codons. The large boxed area
represents the sequence cloned into construct HH-2. Bottom,
schematic illustration of the two LAMC2-LacZ reporter gene
constructs HH-1 and HH-2 used in transgenic mice.
[0023] FIGS. 9A-D. Expression of LAMC2-LacZ reporter gene
constructs in 15.5-day-old transgenic mouse embryos. (9A), Staining
of the whole 15.5-day-old embryo reveals highly restricted
expression of .beta.-galactosidase as observed in some hair
follicles and some regions of skin and testicles (arrow). Construct
HH-2. (9B), Scattered epithelial cells of hair follicles are
positive. Construct HH-1. (9C), Scattered positive epithelial cells
of ductus deferens. Construct HH-1. (9D), Some positive epithelial
cells of skin. Construct HH-2.
[0024] FIGS. 10A-D. Expression of shorter LAMC2-LacZ reporter gene
construct HH-2 in adult transgenic mouse tissues. (10A), Regionally
positive epithelial cells of the epidermis (ep) and hair follicles
(arrow). (10B), Scattered positive epithelial cells in ductus
deferens. (10C and 10D), Expression can be seen in some areas of
the gastric mucosa. Note that the expression can be localized to
epithelial cells of both the surface and in the gastric pits.
[0025] FIGS. 11A-D. Immunolocalization of laminin-5 .gamma.2 chain
and expression of LAMC2-LacZ reporter gene constructs in dorsal
incision skin wounds in tranzsgenic mice. (11A), Immunostaining
localizes the .gamma.2 chain to the basement membrane (arrow)
beneath keratinocytes of the uninjured epithelium (ep) at the edges
of the 3day-old wound. (11B), Immunostaining reveals the presence
of the .gamma.2 chain in the entire newly formed basement membrane
deposited under the epithelium (ep) covering the 7-day-old incision
wound. (11C), Expression of the LAMC2-LacZ reporter gene construct
HH-2 in keratinocytes migrating from edges of the 3-day-old
incision wound (wtaws) during the initial phase of
re-epithelialization. Only minor expression is observed in
keratinocytes of the uninjured epithelium. (11D), Keratinocytes of
the new epithelium covering the entire 7-day-old incision wound
exhibit intense expression of the reporter gene.
DETAILED DESCRIPTION OF THE INVENTION
[0026] Epidermolysis bullosa (EB) is a group of mechano-bullous
disorders characterized by fragility of the skin and mucous
membranes (see Lin & Carter eds., Epidermolysis bullosa. Basic
and clinical aspects, 1992, Springer Verlag, N.Y.; Fine et al., J.
Am. Acad. Dermatol., 1991, 24:119-135). The junctional forms of EB
(JEB) are characterized by tissue separation at the level of the
lamina lucida within the dermal-epidermal basement membrane, and no
specific mutation had yet to be reported. Recently it has been
proposed that the genes for a lamina lucida protein
kalinin/nicein/epiligin may be a candidate in some forms of JEB
(Verrando et al., 1991, supra). Several lines of evidence suggest
that anchoring filament proteins could be defective in some forms
of JEB. First, attenuation or absence of immunoreactivity with
anti-kalinin(epiligrin) antibodies has been noted in the skin of
patients with the most severe (Herlitz) type of JEB. The
immunofluorescence staining patterns may be of prognostic value in
classifying JEB, and these immunoreagents have been used for
prenatal diagnosis of JEB using fetal skin biopsy specimens.
Second, the kalinin/laminin 5 .gamma.-2 chain is expressed in
epithelial cells of the skin, trachea and kidneys, tissues which
are frequently affected by JEB.
[0027] Since the majority of cases are of the generalized (Herlitz)
phenotype (H-5 JEB), JEB patients have been classified into Herlitz
and non-Herlitz types. Clinical features of H-JEB include
mechanical fragility of the skin, with widespread blistering and
erosions, rapid deterioration and neonatal death, often from
sepsis. Longterm survival is rare.
[0028] Efforts to identify the basic defect in JEB began with the
observation that a monoclonal antibody that binds to the lamina
lucida of the epidermal basement membrane zone of normal skin,
fails to react with the lamina lucida of H-JEB skin (Verrando-et
al., 1991, supra.). The antigen recognized by this antibody was
purified from keratinocyte culture medium and termed BM600/nicein.
Keratinocytes cultured from the skin of H-JEB patients attach
poorly to substrate and fail to accumulate immunologically
detectable nicein. Further experiments with antibodies specific for
the .alpha.-3 chain of nicein, demonstrated that they were capable
of inducing the rounding and detachment of adherent keratinocytes
without affecting fibroblasts (Rousselle et al., 1991, supra.).
Thus the correlation in vivo and in vitro of the dermoepidermal
separation with deficient nicein/kalinin/laminin 5 immunoreactivity
and the separation induced by anti-nicein antibody have made the
genes encoding this protein strong candidates for the site of H-JEB
mutations.
[0029] The importance of the .gamma.-2 chain of
nicein/kalinin/laminin 5 in JEB, and epithelial tissues prompted
the investigation into the role such adhesion contacts between
epithelial cells may play in abberant cells. Of primary interest
was the role .gamma.-2 chain of nicein/kalinin/laminin 5 abberant
expression may play in cancer tissue, and a possible role in cancer
dissemination.
[0030] It has been recently shown that in colon adenocarcinoma, a
significant positive correlation between the degree of tumor
budding and the recurrence of tumors following curative surgery
exists, and that this fact is likely to reflect a higher invasive
potential of budding cancer cells as compared with cancer cells
located deeper in the tumor (Hase et al., Dis. Colon Rectum, 1993,
36:627-635). Therefore, as demonstrated in Example 3 below, the
instant invention allows for the useful prognostic determination of
success of surgery, means for monitoring progression of rumor
budding and subsequent prognosis.
[0031] The identification of the role of .gamma.-2 chain allows for
the novel use of kalinin/laminin 5 .gamma.-2 chain and its ligand,
as diagnostic probes of the tumor cell/basement membrane adhesion
interface that is crucial for the invasion of non-malignant
tissues, and identifies invasive cells.
[0032] Thus the identification of the role of .gamma.-2 chain
allows for the novel therapeutic intervention of binding of
kalinin/laminin 5 to its ligand, and thereby reducing the tumor
cell/basement membrane adhesion that is crucial for the invasion of
non-malignant tissues, and method for inhibiting the budding of
tumor masses, and a means for determining the level of .gamma.-2
chain expression as a measure of budding activity of a given
tumor.
[0033] As demonstrated in Example 3 below, the .gamma.-2 chain of
kalinin/laminin 5 is preferentially expressed by invasively growing
malignant cells in human carcinomas. Furthermore, migrating
keratinocytes in wound healing also expressed this gene, pointing
to a role of .gamma.-2 chain in epithelial cell migration both in
malignant and in nonmalignant pathological conditions. The
consistent expression of the .gamma.-2 chain gene in invading
cancer cells reflects a functional importance of this molecule in
vivo in establishing contacts between the invading malignant cells
and a provisional matrix in the immediate surroundings of the
cancer cells. The instant invention provides methods for the
identification of, and diagnosis of invasive cells and tissues, and
for the monitoring of the progress of therapeutic treatments.
[0034] In a preferred embodiment of this aspect of the instant
invention the nucleic acid probe comprise a specifically
hybridizing fragment of the .gamma.-2 chain cDNA nucleic acid
sequence. In this embodiment, the nucleic acid sequence comprises
all or a specifically hybridizing fragment of an open reading frame
of the nucleic acid sequence for the .gamma.-2 chain (FIG. 4)
encoding the amino acid sequence of the .gamma.-2 chain (FIG. 4).
It will be understood that the term "specifically hybridizing" when
used to describe a fragment of nucleic acid encoding a human
laminin .gamma.-2 chain gene is intended to mean that, nucleic acid
hybridization of such a fragment is stable under high stringency
conditions of hybridization and washing as the term "high
stringency" would be understood by those having skill in the
molecular biological arts.
[0035] Further, the instant invention provides for the therapeutic
treatment of such invasive tissues by using .gamma.-2 chain or
biologically active fragments thereof to interfere with the
interactions between abberant .gamma.-2 chain and surrounding
tissues. The instant invention also provides for the intervention
of .gamma.-2 chain interaction with surrounding tissues by using
specific anti-.gamma.-2 chain antibodies (monoclonal or polyclonal)
to inhibit the .gamma.-2 chain biological activity.
[0036] The instant disclosure also allows one to ablate the
invasive cell phenotypic .gamma.-2 chain expression by using
genetic manipulation to "knock-out" the functional expression of
the .gamma.-2 chain gene in cancer cells, or to completely
"knock-out" the functional .gamma.-2 chain gene in the genome of
cancer cells. Such knock-outs can be accomplished by using genetic
molecular biological techniques for inserting by homologous
recombination into genomic DNA, targeted transposon insertion, or
random insertion/deletion mutations in the genomic DNA.
[0037] The instant disclosure also allows for the therapeutic
treatment of invasive cell phenotype by the inhibition of
functional .gamma.-2 chain expression in targeted cells by using
anti-sense technology, such methods for anti-sense production,
stabilization, delivery, and therapeutic approaches are reviewed in
Uhlmann et al., 1990, Chem. Reviews 90:543-584).
[0038] Moreover, the present invention is also directed to two
important functional aspects of the epithelium specific laminin-5,
i.e. cell adhesion and migration. First of all, according to the
present invention, the .gamma.2 subunit chain, as such, does not
promote cell adhesion and, secondly, the laminin-5 isoform and its
.gamma.2 chain subunit play a role in the migratory process of
cells of epithelial origin. According to the present invention, the
migratory function of the .gamma.2 chain is a characteristic for
domain III, as shown with antibody inhibition studies. Furthermore,
involvement of the .gamma.2 chain in cell migration was shown to be
related with a cis-acting element in LAMC2 gene, as studied in
transgenic mice using promoter-reporter gene constructs.
[0039] Thus the instant invention provides for a method of
detection, diagnosis, prognosis, monitoring, and therapeutic
treatment of invasive cell phenotypes.
[0040] The examples below are meant by way of illustration, and are
not meant to be limiting as to the scope of the insant
disclosure.
EXAMPLE 1
Mutation in the .gamma.2 Chain Gene LAMC2 is Critical in Some Cases
of JEB
[0041] A unique scanning strategy using RT-PCR amplification of
LAMC2 sequences was devised to detect truncated forms of .gamma.-2
chain gene transcripts (Pulkkinen et al., Nature Genetics, 1994,
6:293-298). The 3.6 kilobase coding sequence of the LAMC2 mRNA, was
reverse transcribed and amplified with eight pairs of primers,
producing overlapping PCR amplimers designated A-H. The PCR
products were then examined by agarose gel electrophoresis,
followed by MDE heteroduplex analysis. If bands with altered
mobility were detected, the PCR products were sequenced, and
compared with normal sequences from unaffected family members or
unrelated individuals. Intron/exon borders were identified by PCR
analysis of genomic DNA, deduced by comparison with cDNA
sequences.
A Point Mutation Produces Exon Skipping
[0042] When a panel of five unrelated JEB patients were analysed,
the primers used to amplify segment C (nt 1046-1537) produced a
markedly shortened band of 273 base pairs, as compared with the
normal 491 base pairs. No evidence of the normal sized band was
noted, suggesting that the patient was homozygous for this allele.
Direct sequencing revealed that the shortened product resulted from
the deletion of 219 base pairs corresponding to nucleotides
1184-1402 in the cDNA, thus exon 9 was deleted. The remaining
rucleotide sequences within this and other PCR products did not
reveal any additional mutations upon MDE analysis.
[0043] Subsequent examination of the genomic DNA revealed that the
sequences for exons 8, 9 and 10 were present, however a homozygous
G for A substitution at the 3' acceptor splice site at the junction
of intron 8 and exon 9, abolished the obligatory splice site
sequence (AG).
[0044] Examination of another patient revealed that PCR product F
(nt 2248-2777) corresponding to domains I and II of the .gamma.-2
chain, was a band with altered mobility. Sequencing the abnormal
product revealed a 20 bp deletion, followed by a single base pair
(G) insertion in the coding region corresponding to exon 16. This
mutation causes a frameshift which results in a premature stop
codon 51 base pairs downstream from the deletion-insertion,
producing a truncated kalinin/laminin 5 .gamma.-2 chain terminating
at residue 830.
RT-PCR and MDE Analyses
[0045] RNA isolated from fibroblast cell cultures of JEB patients
was used as template for RT-PCR of the LAMC2 mRNA (Epidermal
keratinocytes can also be used). cDNA was prepared from 50 .mu.g of
total RNA in a volume of 100 .mu.L according to manufacturer's
reccomendations (BRL), and oligonucleotide primers were synthesized
on the basis of the cDNA sequence (FIG. 4; Kallunki et al., 1992,
supra.), to generate about 500 base pair products, which spanned
the entire coding region.
[0046] For PCR amplification, 1 .mu.L of cDNA was used as template
and amplification conditions-were 94.degree. C. for 5 min followed
by 95.degree. C. for 45 sec, 60.degree. C. for 45 sec and
72.degree. C. for 45 sec for 35 cycles in an OmniGene thermal
cycler (Marsh Scientific). Amplification was performed in a total
volume of 25 .mu.L containing 1.5 mM MgCl.sub.2, and 2 U Taq
polymerase (Boehringer Mannheim). Aliquots of 5 .mu.L were analysed
on 2% agarose gels and MDE heteroduplex analysis was performed
according to the manufacturer's recommendation (AT Biochemicals).
Heteroduplexes were visualized by staining with ethidium bromide.
If a band of altered mobility was detected in heteroduplex
analysis, the PCR product was subcloned into the TA vector
(Invitrogen), and sequenced by standard techniques.
[0047] DNA isolated either from fibroblast cultures or from
specimens obtained from buccal smears, was used as template for
amplification of genomic sequences. For amplification of introns 8
and 16, about 0.500 ng of genomic DNA was used as template and the
following oligomer primers were utilized. TABLE-US-00003 5'
GGCTCACCAAGACTTACACA 3'; (SEQ ID NO:1) 5' GAATCACTGAGCAGCTGAAC 3';
(SEQ ID NO:2 5' CAGTACCAGAACCGAGTTCG 3'; (SEQ ID NO:3) 5'
CTGGTTACCAGGCTTGAGAG 3'; (SEQ ID NO:4) 5' TTACTGCGGAATCTCACAGC 3';
(SEQ ED NO:5) 5' TACACTGTTCAACCCAGGGT 3'; (SEQ ID NO:6) 5'
AAACAAGCCCTCTCACTGGT 3'; (SEQ ID NO:7) 5' GCGGAGACTGTGCTGATAAG 3';
(SEQ ID NO:8) 5' CATAGCTGTCTACATGGCAT 3'; (SEQ ID NO:9) 5'
AGTCTCGCTGAATCTCTCTT 3'; (SEQ ID NO:10) 5' TTACAACTAGCATGGTGCCC 3';
(SEQ ID NO:11)
Amplification conditions were 94.degree. C. for 7 min followed by
95.degree. C. for 1.5 min, 56.degree. C. (intron 8) or 58.degree.
C. (intron 16) for 1 min and 72.degree. C. for 1.5 min for 35
cycles in an OmniGene thermal cycler (Marsh Scientific).
Amplification was performed in a total volume of 25 .mu.L
containing 1.5 mM MgCl.sub.2, and 2 U Taq polymerase (Boehringer
Mannheim). The PCR products were subcloned and sequenced as above.
Verification of Mutations
[0048] The putative mutations detected in the PCR products were
verified at the genomic level in both cases. For this purpose, a
search for a potential change in restriction endonuclease sites as
a result of the mutation was performed.
[0049] Amplification conditions were 94.degree. C. for 7 min
followed by 94.degree. C. for 1 min. 58.degree. C. for 45 sec and
72.degree. C. for 45 sec for 35 cycles in an OmniGene thermal
cycler (Marsh Scientific). PCR products were analysed on 2.5%
agarose gels.
[0050] The methods described allow for the screening of patients
for mutations in the .gamma.-2 chain which will correlate with JEB.
As demonstrated, the results have identified a homozygous point
mutation resulting in oxon kipping, and a heterozygous
deletion-insertion mutation. This demonstrating the effective
screening for, and identification of, .gamma.-2 chain mutations
which correlate with JEB. The methods are thus useful for
diagnosis, prenatal screening, early screening and detection, as
well as detailed examination of JEB. Further, the results show that
the functional role of .gamma.-2 chain expression in epithelial
cells is important in determining proper intercellular
connectivity, relating to the integrity of tissues and cell
interactions.
EXAMPLE 2
Mutation in the 65 2 Chain Gene LAMC2 is Critical in H-JEB
[0051] The correlation both in vivo and in vitro of the
dermo-epidermal separation in H-JEB, with deficient
immunoreactivity of anti-nicein/kalinin/laminin 5 antibodies, and
the separation induced by anti-nicein/kalinin/laminin 5 antibodies
have made the genes encoding this protein strong candidates for the
site of H-JEB mutations. In this example, it is demonstrated that
the molecular defect which causes H-JEB is linked to the gene
encoding nicein/kalinin/laminin 5 .gamma.-2 chain. In particular,
the occurence of a homozygous premature termination codon mutation
is the specific cause in an examined case of H-JEB (Aberdam et al.,
Nature Genetics, 1994, 6:299-304).
[0052] Expression of mRNA encoding the three nicein subunits by
northern analysis of RNA isolated from primary keratinocyte culture
of a H-JEB patient was determined as the initial screen
Hybridization with probes for the .alpha.-3 and .beta.-3 subunits
was normal, but no hybridization with a cDNA encoding the .gamma.-2
subunit was detected. Examination of the genomic DNA for gross
abnormalities, such as large deletions, insertions or
rearrangements, in LAMC2 (the .gamma.-2 subunit gene) by Southern
blot analysis turned up no abnormalities when the genomic DNA was
digested with BamHI, BgII, HindIII, PstI or PvuII and probed with
full length LAMC2 cDNA.
[0053] Possible mutations in the .gamma.-2 subunit were sought by
using cDNA reverse transcribed from total RNA purified from
cultured keratinocytes of the H-JEB patient, and subjected to PCR
amplification. The size of the amplified products was checked by
electrophoresis on 2% agarose gels and compared with that obtained
from healthy controls.
[0054] No major differences were detected inrthe agarose gels, and
the PCR products were examined by heteroduplex analysis (MDE).
Heteroduplex analysis of the most 5' PCR product (nt 35-726)
revealed the presence of a homoduplex in the proband (pateint) and
the controls. However, when the amplified PCR products from the
patient and control were mixed together, an additional band with
altered mobility, representing heteroduplexes, was detected,
suggesting a homozygous mutation in the patient's LAMC2 cDNA. This
amplified fragment corresponded to domain V of the .gamma.-2
protein (Vailly et al., Eur. J. Biochem., 1994, 219:209-218).
Sequencing detected a C to T transition at position +283, leading
to a nonsense mutation in which a termination codon TGA replaces an
arginine (CGA), perhaps arising as a result of the hypermutability
of 5-methyl-cytosine to thymine at CpG nucleotides. This mutation,
R95X, leads to truncation of the .gamma.-2 subunit polypeptide at
amino acid 95 and loss of a TaqI restriction site (TCGA). Digestion
of cDNA with TaqI confirmed the presence of a homozygous mutation
in the DNA of the H-JEB patient. No other mutations were
detected.
[0055] To confirm the cosegregation of the mutation with the loss
of the TaqI restriction site, eight genotyped individuals of the
family of the patient were screened. In each case, a 120 base pair
fragment was amplified by PCR using genomic DNA templates and
primers flanking the restriction site. Upon digestion of the wild
type amplification product, two cleavage fragments of 80 and 40
base pairs are generated. Consistent with the presence of a
heterozygous mutation in carriers of this genotype, DNA fragments
of 120, 80 and 40 base pairs, indicative of a wild type genotype,
were found in the paternal grandmother and two other relatives.
Cell Culture
[0056] Epidermis was separated from dermis by dispase treatment at
37.degree. C. Keratinocytes were dissociated in 0.25% trypsin at
37.degree. C. and plated onto a feeder layer of irradiated mouse
3T3 cells (ICN) (Rheinwald & Green, Cell, 175, 6:331-334).
Keratinocytes were grown in a 1:1 mixture of DMEM and Ham's F12
(BRL) containing 10% Fetal Calf Serum (FCS), 1 mM sodium pyruvate,
2 mM L-glutamine, 10 .mu.g/mL of penicillin and strptomycin, 10
ng/mL transferrin, 180 .mu.M adenine and 20 pM T3 (Simon &
Green, Cell, 1985, 40:677-683). H-JEB keratinocytes were expanded
after gentle dissociation in 0.05% trypsin, 0.02% EDTA.
Northern Blot Analysis
[0057] Total RNA was prepared from H-JEB and normal cultured
keratinocytes according to standard methods (Chomzynski &
Sacchi, Anal. Biochem., 1987, 162: 156-159). RNA was
electrophoresed in 1.2% denaturing agarose gels containing 1.2 M
formaldehyde and transferred onto Hybond N membrane (Amersham).
Membranes were hybridized at high stringency with P-32 labeled cDNA
probes corresponding to the different chains of nicein, and then
exposed on Hyperfiln MP (Amersham) with intensifying screens.
Radiolabeled cDNA probes NAI (Baudoin et al., J. Invest. Dermatol.,
1994, in press), KAL-5.5 C (Gerecke et al., Eur. J. Biochem., 1994,
in press), and PCR 1.3 (Vailly et al., 1994, supra), were used to
detect the mRNAs for nicein chains .alpha.-3, .beta.-3 and
.gamma.-2, respectively.
RT-PCR and Heteroduplex Analysis (MDE)
[0058] 50 .mu.g of total RNA isolated from cultured keratinocytes
from JEB patient, and unrelated healthy controls were reverse
transcribed in a volume of 100 .mu.L as recommended by the
manufacturer (BRL). 1 .mu.L of the reaction product was used to
amplify overlapping regions of the cDNA that spanned the open
reading frame. Primer pair used to identify the mutation R95X: (L)
5'-GAGCGCAGAGTGAGAACCAC-3' SEQ ID NO:16, (R)
5'-ACTGTATTCTGCAGAGCTGC-3' SEQ ID NO:17. PCR cycling conditions
were: 94.degree. C., 5 min, followed by 94.degree. C., 45 sec;
60.degree. C., 45 sec; 72.degree. C., 45 sec; for 35 cycles, and
extension at 72.degree. C. for 5 min. 5 .mu.L aliquots were run in
2% agarose gels. Heteroduplex analysis was performed as recommended
by the manufacturer (MDE, AT Biochemicals). Heteroduplexes were
visualized under UV light in the presence of ethidium bromide and
photographed. Amplified cDNA fragments with altered mobility were
subcloned into the TA vector according to the manufacturer's
recommendations (Invitrogen). Sequence analysis were then performed
using standard techniques.
Verification of the Mutation
[0059] PCR reactions on genomic DNA (50 .mu.g) were carried out
using the upstream primer 5'-TTCCTTTCCCCTACCTTGTG-3' (SEQ ID NO:18)
and the downstream printer 5'-TGTGGAAGCCTGGCAGACAT-3' (SEQ ID
NO:19), which are located in the intron 2 and exon 3 of LAMC2
respectively. PCR conditions were: 95.degree. C., 5 min, followed
by 94.degree. C., 45 sec; 56.degree. C., 45 sec; 72.degree. C., 45
sec; for 35 cycles, and extension at 72.degree. C. for 5 min. PCR
products were used for restriction analysis. 20 .mu.L of PCR
product obtained from genomic DNA was digested with TaqI for 2
hours (Boehringer Mannheim). Cleavage products were electrophoresed
(2.4% agarose) stained and visualized under UV light.
[0060] Thus the methods allow for the screening of patients for
mutations in the .gamma.-2 chain which correlate with H-JEB. As
demonstrated, the results have identified a nonsense mutation
resulting in a truncated .gamma.-2 chain, leading to severe H-JEB.
This was further confirmed by specific amplification and
restriction enzyme analysis of both the patient and relatives. Thus
demonstrating the effective screening for, and identification of
.gamma.-2 chain mutations which correlate with H-JEB. The methods
are thus useful for diagnosis, prenatal screening, early screening
and detection, as well as detailed examination of H-JEB.
Furthermore, the results demonstrate the significance of the
.beta.-2 chain in forming proper cellular contacts.
EXAMPLE 3
.gamma.-2 Chain as Diagnostic for Invasive
[0061] In this example, in situ hybridization is used to
demonstrate the expression of the kalinin/laminin 5 .gamma.-2 chain
in a variety of human cancer tissues and in skin wound healing in
mice (Pyke et al., Amer. J. Pathol., Oct. 1994, 145(4): 1-10).
[0062] Thirty-six routinely processed, formalin-fixed and paraffin
wax-embedded specimens from cancer surgery performed from 1991 to
1993 were drawn from pathology department files at Herlev Hospital
(Copenhagen, Denmark). The specimens were evaluated according to
standard criteria and included 16 cases of moderately or well
differentiated colon adenocarcinomas, 7 cases of ductal mammary
carcinomas, 4 squamous cell carcinomas (2 skin, 1 cervix, 1 vulva),
3 malignant melanomas, and 6 sarcomas (3 leiomyosarcomas, 2
malignant fibrous histiocytomas, 1 neurofibrosarcoma).
[0063] All samples were selected upon histological examination of a
hematoxylin and eosinstained section to ensure that they showed a
well preserved morphology throughout and contained representative
areas of both cancerous tissue and surrounding apparently normal,
unaffected tissue. The broad zone separating these two tissue
compartments is referred to as the invasive front in the following.
No estimation of the effect of variations in fixation conditions
was attempted, but in a previous study of plasminogen activating
system components using specimens of colon adenocarcinomas
collected using the same procedures, very little variation in
relative mRNA levels was found (Pyke, C. PhD. Thesis, 1993,
University of Copenhagen, Denmark). In addition, tissue from
incisionally wounded mouse skin prepared as described by Romer et
al. (J. Invest. Dermatol., 1994, 102:519-522), was fixed and
paraffin-embedded the same way as the human cancer specimens.
[0064] For preparation of total RNA from six samples of colon
adenocarcinomas, tissues were snap-frozen in liquid nitrogen
immediately following resection and RNA was prepared as described
by Lund et al., (Biochem. J., 1994).
Probes:
[0065] Fragments of the cDNA for the .gamma.-2 chain of human
kalinin/laminin 5 were inserted into RNA transcription vectors by
restriction enzyme cutting of clone L15 covering base pairs 2995 to
3840 (FIG. 4; Kallunki et al., 1992, supra.). In brief plasmids
phb2t-01 and phb2t-02 were prepared by insertion of the complete
L15 .gamma.-2 chain cDNA in sense and anti-sense orientation into
the polylinker of plasmid vectors SP64 and SP65 (both Promega,
Madison, Wis.), respectively. In addition, two non-overlapping
fragments of clone L15 were bluntend cloned into the EcoRV-site of
pKS(Bluescript)II(+) (Stratagene, La Jolla, Calif.) transcription
vector and the resulting plasmids were verified by dideoxy
sequencing according to Sanger et al. (PNAS(USA), 1977,
74:5463-5471). Plasmid phb2t-03 cover bases 3003-3239 and phb2t-05
cover bases 3239 to 3839, numbers referring to cDNA sequence Z15008
in the EMBL/GenBank/DDBJ database as reported by Kallunki et al.,
(1992, supra; FIG. 4).
[0066] Similarly, cDNA fragments of other human laminin chains were
prepared in RNA transcription vectors, yielding the following
plasmid constructs (numbers in brackets refer to base pair numbers
in the EMBL/GenBank/DDBJ sequence database by the listed accession
numbers); chain .alpha.-1: plasmid phae-01 (3244-3584 (accession
No. X58531, Nissinen et al., Biochem. J., 1991, 276:369-379) in
pKS(Bluescript)II(+)); chain .beta.-1: plasmid phble-01 (3460-4366
(accession No. J02778, Pikkarainen et al., J. Biol. Chem., 1987,
262:10454-10462) in pKS(Bluescript)II(+)); chain .beta.-1: plasmids
A1PSP64 and A1PSP65 (919-1535 (accession No. M55210, Pikkarainen et
al., J. Biol. Chem., 1988, 263:6751-6758) in SP64 and SP65
repectively (sense and anti-sense orientation)).
[0067] All plasmids were linearized for transcription using
restriction endonucleases and 5 .mu.g of the linearized plasmids
was extracted with phenol and with choloroform/isoamyl alcohol
(25:1), precipitated with ethanol, and redissolved in water. Each
transcription reaction contained 1 .mu.g linearized DNA template,
and transcriptions were performed essentially as recommended by the
manufacturer of the polymerases. The RNA was hydrolyzed in 0.1
mol/L sodium carbonate buffer, pH 10.2, containing 10 mmol/L
dithiothreitol (DTT) to an average size of 100 bases. RNA probes
transcribed from opposite strands of the same pIasmid template,
yielding sense and anti-sense transcripts, were adjusted to
.times.10.sup.6 cpm/.mu.L and stored at -20.degree. C. until used.
Probes were applied to tissue sections.
In Situ Hybridization:
[0068] In situ hybridization was performed as described by Pyke et
al., (Am. J. Pathol., 1991, 38:1059-1067) with 35S labeled RNA
probes prepared as described above. In brief, paraffin sections
were cut, placed on gelatinized slides, heated to 60.degree. C. for
30 minutes, deparaffinized in xylene, and rehydrated through graded
alcohols to PBS (0.01 mol/L sodium phosphate buffer, pH 7.4,
containing 0.14 mol/L NaCl). The slides were then washed twice in
PBS, incubated with 5 .mu.g/mL proteinase K in 50 mmol/L Tris/HCl,
pH 8.0, with 5 mmol/L EDTA for 7.5 minutes, washed in PBS (2
minutes), dehydrated in graded ethanols, and air-dried before the
RNA probe (.about.80 pg/.mu.L) was applied. The hybridization
solution consisted of deionized formamide (50%), dextran sulfate
(10%), tRNA (1 .mu.g/.mu.L), Ficoll 400 (0.02% (w/v)),
polyvinylpyrrolidone (0.02% (w/v)), BSA fraction V (0.02% (w/v)),
10 mmol/L DTT, 0.3 M NaCl, 0.5 mmol/L EDTA, 10 mmol/L Tris-HCl, and
10 mmol/L NaPO.sub.4 (pH 6.8). Sections were covered by
alcohol-washed, autoclaved coverslips and hybridized at 47.degree.
C. overnight (16 to 18 hours) in a chamber humidified with 10 ml of
a mixture similar to the hybridization solution, except for the
omission of probe, dextran sulfate, DTT, and tRNA (washing
mixture). After hybridization, slides were washed in washing
mixture for 2.times.1 hour at 50.degree. C., followed by 0.5 mol/L
NaCl, 1 mmol/L EDTA, 10 mmol/L Tris-HCl (pH 7.2) (NTE) with 10
mmol/L DTT at 37.degree. C. for 15 minutes. After treatment with
RNAse A (20 .mu.g/mL) in NTE at 37.degree. C. for 30 minutes, the
sections were washed in NTE at 37.degree. C. (2.times.30 minutes),
and in 2 L of 15 mmol/L sodium chloride, 1.5 mmol/l sodium citrate,
pH 7.0, with 1 mmol/L DTT for 30 minutes at room temperature with
stirring. Sections were then dehydrated and airred. Finally,
autoradiographic emulsion sec applied according to the
manufacturer's reccomendations, and sections were stored in black
airtight boxes at 4 C. until they were developed after 1 to 2 weeks
of exposure.
Resuts; Laminin .alpha.-1, .beta.-1, .gamma.-1, and .gamma.-2
chains
[0069] All rounds of in situ hybridization include both sense and
anti-sense RNA probes for each of the genes studied. As negative
controls, sense RNA probes are applied to adjacent sections and
these probes consistently are negative. As a positive control of
the .gamma.-2 chain hybridizations, two anti-sense probes derived
from non-overlapping .gamma.-2 chain cDNA clones are used on a
number of sections. To summarizes the .gamma.-2 chain expression
found; all carcinomas were positive except for one case of mammary
duct carcinoma, and all three cases of leiomyosarcomas, both cases
of malignant fibrous histiocytoma, and the only case of
neurofibrosarcoma. The positive controls always give similar
staining on adjacent sections (see FIG. 2, E and G). Fifteen of the
malignant cases and all mouse tissue blocks were hybridized on two
or more separate occasions giving the same hybridization pattern.
All cell types other than those described below were negative in
all cases.
Colon Adenocarcinoma
[0070] Sixteen specimens of colon adenocarcinoma were investigated
by in situ hybridization for expression of the .gamma.-2 chain
(FIG. 1). In all of these cases, mRNA for .gamma.-2 chain was
present exclusively in cancer cells and in most of the cases,
staining was confined to a distinct subpopulation of cancer cells
at the invasive front (FIG. 1, A-D). A characteristic feature of
.gamma.-2 chain containing cancer cells at the invasive front was
that they appeared to represent cells in the process of branching
or dissociating from larger well differentiated epithelial glands,
a phenomenon referred to in the literature as tumor budding or
tumor cell dissociation.
[0071] In normal-looking colon mucosa distal from the invasive
carcinoma, moderate signals for .gamma.-2 chain mRNA were observed
in two specimens in the epithelial cells of a few mucosal glands
that showed clear morphological signs of glandular disintegration
and phagocytic cell infiltration Apart from this, a weak signal was
seen in luminal epithelial cells in normal looking colon mucosa in
most specimens.
[0072] Weak signals for laminin chains .alpha.-1, .beta.-1, and
.gamma.-1 mRNAs were detected in cancerous areas of the 6 colon
cancers studied for the expression of these genes. The expression
of each of the three genes showed a similar distribution.
Expression in stromal cells with a fibroblast-like morphology as
well as in endothelial cells of smaller vessels was consistently
found. In marked contrast to the .gamma.-2 chain expression in the
same samples, expression of .alpha.-1, .beta.-1, or .gamma.-1 was
never found in cancer cells and no correlation between expression
of .alpha.-1, .beta.-1, and .gamma.-1 chains with sites of invasion
was found. Adjacent normal-looking parts of the samples were
negative or only weakly positive for these laminin chains.
[0073] FIG. 1 shows in situ hybridization of a specimen of colon
adenocarcinoma for .gamma.-2 chain mRNA using a S-35 labeled
anti-sense RNA probe derived from plasmid pbb2r02. FIG. 1A is a
cluster of heavily labeled cancer cells at the invasive front (open
arrow) in close proximity to a well-differentiated glandular
structure (straight arrow). FIG. 1B shows a high-magnification view
of the area at the open arrow in 1A. Note that the isolated cancer
cells show prominent labeling, whereas many coherent cancer cells
of an adjacent glandular structure are negative (straight arrow).
FIG. 1C shows the same pattern at an invasive focus in another part
of the same specimen. FIG. 1D shows strong .gamma.-2 chain
expression in cancer cells engaged in a bifurcation process (curved
arrows). The malignant glandular epithelium from which the
.gamma.-2 chain-positive cancer cells are branching is negative
(straight arrow). Magnification:1A.times.100; 1B-1D.times.640.
Ductal Mammary Carcinomas
[0074] Six of the seven cases showed a prominent signal for
.gamma.2 chain in a small subpopulation of cells intimately
associated with invasively growing malignant glandular structures.
The most prominent signal was seen in cells located at the border
between malignant and surrounding stromal tissue in glandular
structures that exhibited clear histological signs of active
invasion (FIG. 2A). On careful examination it was concluded that
the majority of the positive cells were cancer cells but it was not
possible to determine if the cells of myoepithelial origin were
also positive in some cases. One case was totally negative.
Normal-appearing glandular tissue was negative in all cases.
[0075] Weak signals for laminin chains .alpha.-1, .beta.-1, and
.gamma.-1 mRNAs were detected in fibroblast-like stromal cells
throughout cancerous areas in one of the cases.
Malignant Melanoma
[0076] In all three cases strong hybridization of .gamma.-2 chain
was found in a population of cancer cells in the radial growth
phase (FIG. 2B). Laminin chains .alpha.-1, .beta.-1, and .gamma.-1
were weakly expressed in the endothelium of small vessels and in
fibroblast-like stromal cells throughout the affected areas in the
two cases studied for these components. In addition, a weak signal
for these chains was seen in sebaceous glands of adjacent normal
skin.
Squamous Cell Carcinomas
[0077] In all four squamous cell carcinomas investigated, the same
pattern of .gamma.-2 chain expression was found as in other
carcinomas. The signals were found only in cancer cells, and only
in areas with signs of ongoing invasion (FIG. 2, C-G).
[0078] The four cases were also studied for mRNA of .alpha.-1,
.beta.-1, and .gamma.-1 chains. In the two skin cancers, it was
found that a very weak signal occurred in malignant cells, and that
the weak signal was in all cancer cells and of an equal intensity.
This is in clear contrast to the pattern of expression of the
.gamma.-2 chain. As seen in melanomas, epithelial cells of
sebaceous glands present in adjacent unaffected skin were weakly
positive for these laminin chains. In the other two cases (cervix
and vulva) weak expression of .alpha.-1, .beta.-1, and .gamma.-1
chains were seen only in endothelial and fibroblast-like stromal
cells throughout the cancerous areas (FIG. 2F).
[0079] FIG. 2 shows In situ hybridization for .gamma.-2 chain mRNA
on sections of ductal mammary carcinoma (2A), malignant melanoma
(2B), squamous cell carcinoma of the skin (2C-2D), and squamous
cell carcinoma of the vulva (2E-2G). In 2A, cancer shows prominent
signal for .gamma.-2 chain mRNA in cells bordering the zone between
malignant glandular tissue and surrounding mesenchyme (curved
arrows). Cancer cells located more centrally in individual
malignant glandular structures are negative for .gamma.-2 chain
mRNA (straight arrows). Note the wedge shaped form of the invading
glandular tissue. (All images marked X' are darkfield images of the
respective sections). FIG. 2B shows .gamma.-2 chain mRNA signal in
a subpopulation of cancer cells of radially growing malignant
epithelium (curved arrows). Adjacent malignant epithelium showing a
different growth pattern is devoid a signal (straight arrow). FIG.
2C shows .gamma.-2 chain mRNA containing cancer cells at the
invasive front (curved arrow). Note lack of signal in non-invasive
areas of the tumor and in adjacent unaffected areas (straight
arrow). FIG. 2D is a higher magnification of area of curved arrow
of 2C highlighting the prominent signal in invading cells (curved
arrow). Adjacent cancer cells with tumor islets are negative
(straight arrow). FIG. 2E shows a strong signal for .gamma.-2 chain
mRNA is seen in invading cancer cells, using an anti-sense RNA
probe derived from plasmid pb2t-03 (curved arrow). A-postcapillary
venule is negative (straight arrow). FIG. 2F is a near adjacent
section hybridized for laminin .gamma.-1 chain. Note that the
endothelial cells of the venule show signal (straight arrow)
whereas the malignant epithelium is negative (curved arrow). FIG.
2G is another near-adjacent section which was hybridized for
.gamma.-2 chain expression using an anti-sense RNA probe derived
from a cDNA plasmid non-overlapping with that used for preparing
the probe in 2E (phb2t-05). Note that the hybridization pattern is
similar to that seen in 2E, with strong signal in invading cancer
cells (curved arrow) and absence of signal in a vessel (straight
arrow). Magnification: 2C.times.100, all others.times.640.
Sarcomas
[0080] All six sarcomas tested in the study were totally negative
for .gamma.-2 chain mRNA. The expression of other laminin chains
was not tested.
Mouse Wounded Skin
[0081] To compare the gene expression of .gamma.-2 chain in cancer
tissue with a nonmalignant condition known to contain actively
migrating epithelial cells showing a transient invasive phenotype,
we hybridized sections of incisionally wounded mouse skin with
.gamma.-2 chain sense and anti-sense RNA probes. Weak .gamma.-2
chain expression was observed in the keratinocytes at the edge of
12-hour old wounds, and at later time points (1-5 days), strong
signals for .gamma.-2 chain mRNA was seen exclusively in the basal
keratinocytes of the epiderral tongue moving under the wound clot
(FIG. 3). In adjacent normal-looking skin, keratinocytes were
negative for .gamma.-2 chain mRNA.
[0082] FIG. 3 is incisionally wounded mouse skin (.gamma.-2 hours
after wounding) showing signal for .gamma.-2 chain in keratinocytes
at the leading edge of the migrating epithelium (curved arrow).
Whereas buccal keratinocytes located more distant to the site of
injury show little or no signal (straight arrow). Note that the
signal for .gamma.-2 chain stops at the tip of invading
keratinocytes (open arrow). A' is a dark field image of 3A.
Magnification: .times.640.
RNAse Protection Assay
[0083] Plasmid phbt-03 was linearized with EcoRI and a radiolabeled
RNA-anti-sense probe was prepared by transcription using .sup.32 P
UTP and T3 polymerase (Pyke et al., FEBS Letters, 1993, 326:69-75).
RNAse protection assay, using 40 .mu.g ethanol-precipitated and
DNAse .mu.I-treated total RNA from six samples of colon
adenocarcinomas was performed as described in Pyke et al., (1993,
supra.). Protected mRNA regions were analyzed on a denaturing
polyacrylamide gel and autoradiography.
[0084] The RNAse protection assay carried out on total RNA from the
six samples confirmed the presence of genuine .gamma.-2 chain mRNA
in all samples.
[0085] These results clearly demonstrate the important correlation
of .gamma.-2 chain expression and invasive cell phenotype in vivo,
as detected in vitro. Thus the instant methods present a novel and
important method for the specific identification of invasive cell
phenotypes in biopsied tissues. The knowledge of any information
diagnostic for the presence or absence of invasive cells is useful
for the monitoring and prognosis of continuing anti-carcinoma
therapies. Further the identification of the expression or
non-expression of the .gamma.-2 chain provides important
information as to the phenotypic nature of the tissue examined.
Thus the instant example demonstrates the use of probes of
.gamma.-2 chain for detection of the presence, or absence, of
invasive cells.
EXAMPLE 4
[0086] The following example demonstrates the functional aspects of
laminin-5, including the .gamma.-2 chain of laminin-5, on cell
adhesion and cell migration.
Materials and Methods
[0087] Cells and Cell Culture--A mouse squamous cell carcinoma cell
line, KLN-205 (cat. no. ATCC CRL-1453), was obtained from American
Type Culture Collection (Rockville, Md.). The cells were maintained
as monolayer cultures in Eagle's minimum essential medium
(containing non-essential amino acids and Earle's BSS supplemented
with 10% fetal calf serum (FCS). The HaCat human keratinocyte cell
line was a kind gift from Dr. Fuzenig (Heidelberg, Germany). The
HaCat cells were cultured in Dulbecco's MEM supplemented with 10%
FCS. However, when the cells were cultured for the production of
laminin-5, the medium was replaced by serum-free medium.
[0088] Preparation of Proteins--Mouse EHS laminin (laminin-1) was
obtained from GIBCO BRL. Fibronectin was purified from FCS using a
gelatin-Sepharose 4B column (Sigma) as described elsewhere (Vuento,
M. & Vaheri, A. (1979) Biochem. J. 183: 331-337.34. Gillies, R.
J., Didier, N. & Denton, M. (1986) Anal. Biochem. 159:
109-113). Human laminin-5 was immunoaffmity purified from the media
of HaCat cells cultured for three days in the absence of serum.
Briefly, the medium was first passed through a 5 ml
gelatin-Sepharose column (Sigma, St. Louis, Mo.) to ensure the
complete absence of fibronectin from the protein preparation, after
which the medium was passed through a 10 ml anti-laminin
.gamma.2-Sepharose affinity column in order to bind laminin-5
molecules. Both columns were equilibrated in phosphate-buffered
saline. The anti-laminin .gamma.2-Sepharose affinity column was
prepared by coupling a Protein A-purified anti-.gamma.2 IgG (8
mg/ml) to 10 ml of CNBr-activated Sepharose (Pharmacia, Uppsala,
Sweden). The anti-.gamma.2 IgG was purified from a rabbit
polyclonal antiserum prepared against a GST-fusion protein
containing part of the short arm (domain III) of the .gamma.2 chain
(Pyke, C., Salo, S., Ralfkiaer, E., Romer, J., Dano, K. &
Tryggvason, K. (1995) Cancer Res. 55: 4132-4139). The laminin-5 was
eluted from the immunoaffinity column using 50 mM tiethanolamine,
pH 11.25, 0.1% Triton X-100 and neutralized directly with 1 M
Tris-HCl, pH 7.0. Collected fractions were analyzed by SDS-PAGE and
Western blotting using the same polyclonal antibodies as used for
the preparation of the affinity column. Fractions containing
laminin-5 were pooled and dialyzed against 50 mM Tris-HCl, 0,1 M
NaCl, pH 7.4. Some batches of laminin-5 were denatured with 5 M
urea and renatured to study the effects of the treatment on
adhesion and migration properties.
[0089] Generation of Recombinant Baculovirus and Expression of
Recombinant Laminin .gamma.2 Chain--The .gamma.2 chain of laminin-5
was expressed as recombinant protein using the baculovirus system
and purified for studies on its functional properties. A
full-length human laminin .gamma.2 chain cDNA containing L52 UTR
and 822 bp of the 3' UTR was constructed from fbur overlapping cDNA
clones L52, HT2-7, L15 and L61 (Kallunki, P., Sainio, K., Eddy, R.,
Byers, M., Kallunki, T., Sariola, H., Beck, K., Hirvonen, H, Shows,
T. B. & Tryggvason, K. (1992) J. Cell Biol. 119: 679-693). The
resulting 4,402 bp cDNA was analyzed by restriction enzyme mapping
and partial sequencing, and cloned into the pVL1393 recombinant
transfer plasmid prior to transfer into the AcNPV-.gamma.2
baculovinis vector kindly provided by Max Summers (Texas A&M
University). This baculovirus vector containing the human laminin
.gamma.2 chain cDNA under the transcriptional control of the
polyhedrin promoter was produced and purified following standard
procedures (Summers, M. D. & Smith, G. E. (1987). A manual of
15 methods for baculovirus vectors and insect cell culture
procedures. Texas agricultural experiment station bulletin no.
1555, Collage Station, Tex.), except that it was first enriched
according to the method of Pen et al. (Pen, J., Welling, G. W.
& Welling-Wester, S. (1989). Nucl. Acid. Res. 17: 451) from the
virus containing medium obtained by co-transfecting Sf9 cells with
the wild-type virus (AcNPV) DNA and the recombinant transfer vector
pVL1393-.gamma.2. For expression of the recombinant protein, High
Five (H5) cells were infected with the recombinant virus at a
multiplicity of infection (MOI) of 5-10 pfu per cell by using the
standard protocols (Summers, M. D. & Smith, G. E. (1987)).
[0090] The recombinant .gamma.2 chain was purified by first
resuspending the cells in 10 volumes of 50 mM Tris-HCl, pH 7.4, 100
mM NaCl, 2.5 mM EDTA, 1% Triton X-100, 1 mM PMSF and 1 mM NEM
followed by homogenization in a Dounce homogenizer. The protein was
extracted for 60 min on ice and solubilized proteins were removed
by centrifgation at 1500.times.g for 10 min at 4.degree. C. The
pellet was extracted again with buffer containing 1-3 M urea. The
recombinant .gamma.2 chain was extracted with a buffer containing 5
M urea, and renatured by dialysis against 50 mM Tris-HCl, pH 7.4,
100 mM NaCl.
[0091] Preparation of Antibodies--Polyclonal antiserum against
domain III of the laminin .gamma.2 chain was prepared and
characterized as described previously. Briefly, rabbits were
immunized s.c. four times using a .gamma.2-GST fusion protein as
antigen. The antigen contained 177 amino acid residues (res. #
391-567) from domain III of the .gamma.2 (Kallunki, P., Sainio, K.,
Eddy, R., Byers, M., Kallunki, T., Sariola, H., Beck, K., Hirvonen,
H., Shows, T. B. & Tryggvason, K. (1992) J. Cell Biol. 119:
679-693). Antibodies against the GST-epitopes were removed from the
antisera by negative immunoadsorption with GST-Sepharose made by
coupling E. coil expressed GST protein to CNBr-activated Sepharore.
The removal of anti-GST IgG was ensured by Western blotting
analysis with GST-specific antibodies (data not shown). The
specificity of the antibody against the laminin .gamma.2 chain was
also tested by Western blotting as well as by ELISA.
[0092] Polyclonal antibody against the C-terminus of the laminin
.gamma.2 chain was produced in rabbits essentially as above for
domain III using a .gamma.2-GST fusion protein as antigen. The
antigen contained 161 amino acids (res. # 1017-1178) from domain
I/II of the .gamma.2 chain and antibodies against the GST-epitopes
were removed from the antisera by negative immunoadsorption with
GST-Sepharose. The specificity of the antibody was tested by
Western blotting and ELISA.
[0093] Polyclonal antiserum against laminin-1 was a kind gift of
Dr. Foidart (University of Liege, Belgium). Normal rabbit serum was
obtained prior to immunization from the rabbits used for
immunization. IgG from the laminin-1 and laminin .gamma.2 chain
antisera, as well as from normal rabbit serum, was purified using
Protein A Sepharose (Pharrnacia, Uppsala, Sweden).
[0094] Cell Ahdesion Assay--Microtiter plates (96 wells: Nunc,
Copenhagen, Denmark) were coated with 100 .mu.l/well of of
laminin-1 (10 .mu.g/ml), laminin-5 (10 .mu.g/ml), or recombinant
laminin .gamma.2 chain (10 .mu.g/ml) in PBS or 50 mM Tris-HCl, pH
7.4 by incubating the plates overnight at 4.degree. C. Control
wells were uncoated or coated with the same amounts of BSA In some
experiment the proteins were first denatured by dialysis overnight
against 5 M urea, 50 mM Tris-HCl, pH 7.5 and then renatured by
dialysis against 50 mM Tris-HCl, pH 7.5. Potential remaining active
sites on the plates were blocked with 150 .mu.l of 10 mg/ml BSA in
PBS for 2 hours at room temperature. The wells were washed with
PBS, and 100 ml of Eagle's MEM containing 5 mg/ml BSA was added.
For the adhesion assays, KLN-205 cells were detached from
subconfluent cell culture dishes with trypsin-EDTA (0.25%-0.03%)
and resuspended in Eagle's MEM/BSA (5 mg/ml) at a concentration of
2.times.105 cells/ml and allowed to recover for 20 min at
37.degree. C. A total of 20,000 cells were then added to each well
and allowed to attach for additional 90 min at 37.degree. C. The
extent of cell adhesion was determined by measuring color yields at
600 nm, following fixation with 3% paraformaldehyde and staining
with 0.1% crystal violet (Gillies, R. J., Didier, N. & Denton,
M. (1986) Anal. Biochem. 159: 109-113). For inhibition assays with
the anti-.gamma.2 antibody, the substrate coated wells were
incubated with 20 .mu.g/ml of anti-.gamma.2 chain IgG in PBS for 60
minutes prior to incubations with the cells.
[0095] Migration-assay--The effect of endogenous laminin-5 on
migration of KLN-205 cells was determined by using a modified
Boyden chamber assay, as described by Hujanen and Terranova
(Hujanen, E. & Terranova, V. P. (1985) Cancer Res. 45:
3517-3521), and the effect of exogenous laminin-5 by using a
modified Transwell assay, as described by Pelletier et al.
(Pelletier, A. J., Kunicki, T. and Quaranta, V. (1996). J Biol Chem
271: 1364).
[0096] The Boyden chamber assay was briefly carried out as follows.
Polycarbonate filters (pore size 10 .mu.m, diameter 12 mm; Costar,
Cambridge, Mass.) were coated with 2.5 .mu.g of EHS type IV
collagen, and used to separate the upper and lower compartments of
the 50 .mu.l chamber. A total of 1.times.10.sup.5 cells in Eagle's
MEM containing 0.1% BSA were placed in the upper compartment, and
the lower compartment was filled with medium with or without
chemoattractants (50 .mu.g/ml laminin-1 or fibronectin). To study
the effect of the laminin .gamma.2 chain antibodies on cell
migration, anti-.gamma.2 (III) IgG or anti-.gamma.2 (C-term) IgG
was added to the upper compartment together with the cells at a
concentration 20 .mu.g/ml. Normal rabbit IgG was used as negative
control. After 8 hour incubation at 37.degree. C. in a humidified
atmosphere, the filters were removed, fixed and stained
(Diff-Quick, Baxter Diagnostics, Tubingen, Germany). The cells that
had not migrated were removed from the upper surface of the filter
with cotton swabs. Migration of cells was quantified by counting
the cells on the lower surface of each filter in 10 randomly
selected high power fields (.times.400). All-assays were performed
in triplicates.
[0097] The "Transwell" plate assay (Tranwell plates with pore size
12 .mu.m, diameter 12 mm; Costar, Cambridge, Mass.) was used to
determine the effect of exogenous laminin-5 on cell migration. The
lower side of the membrane was coated with 2.5 .mu.g of EHS type IV
collagen for 3 hours at room temperature. Both side were blocked
with 1% bovine serum albumin for 1 hour. A total of
1.times.10.sup.5 cells were added per well in the upper compartment
in Eagle's MEM containing 10% FCS, and the lower compartment was
filled with 2.5 .mu.g/ml laminin-5 as a chemoattractant. Antibodies
against the C-terminus and domain III of the .gamma.2 chains or
nonimmune IgG were added to the upper compartment, together with
the cells at a concentration 20 .mu.g/ml. Following a 16 hour
incubation at 37.degree. C. the cells were fixed and stained with
Diff-Quick. Cells on the top surface of the membrane were removed
with cotton swabs, and cells that had migrated to the lower side of
the membrane were counted (12 fields +/- S.D.)
[0098] Preparation of LAMC2-lacZ Reporter Gene Constructs--Two
different segments of the LAMC2 5' flanking regions were subcloned
into a pKK2480 vector containing the .beta.-galactosidase gene and
the SV40 polyadenylation signal (kind gift from Mikkel Rohde,
Aarhus, Denmark) for expression in transgenic mice. The longer
construct, pHH-1, contained 5946 base pairs (-5,900 to +46) and the
shorter construct contained a total of 668 base pairs (-613 to
+55). Briefly, the 5' end of the pHH-1 insert was made by ligating
a 3,900 base pair HindIII-PstI fragment and a 1,150 base pair PstI
fragment of genomic clone P14 (Airenne, T., Haakana, H., Sainio,
K., Kallunki, T., Kallunki, P., Sariola, H. & Tryggvason, K.
(1996) Genomics 32: 54-64). The 3' end of the construct was a
PstI-SA1I fragment (-699 to +46) made by PCR and ligated to the 5'
end 5,050 base pair fragment. The full length fragment was blunt
ended with Klenow and subcloned into the pKK2480 vector. The
shorter construct pHH-2 was made by PCR from genomic clone P14,
digested with SalI and XbaI and subcloned into pKK2480. All PCR
made segments were sequenced to ensure that no sequence errors
existed. Both constructs could be released from the vector with
XhoI and EagI.
[0099] Generation and Analyses of Transgenic Mice--The plasmids
pHH-1 and pHH-2 were digested with EagI and XhoI to release the
inserts from the vector. Transgenic mice were produced by
pronuclear microinjection of (C57B1/6+DBA/2)F1 fertilized oozytes
as described elsewhere (Hogan, B., Corstantni F. & Lacy, E.
(1986) Manipulating the mouse embryo: A Laboratory Manual. Cold
Spring Harbor, N.Y.). Founder animals were identified by PCR
analysis (Hanley, T. & Merlie, J. P. (1991) BioTechiniques 10:
56) or Southern blotting of genomic DNA isolated from the tail.
Positive founder mice were mated with wild-type hybrid
(C57B1/6+DBA/2)F1 mice to yield transgenic lines. Expression of the
lacZ gene was detected by staining with X-gal
(5bromo4-chloro-3-indolyl-b-D-galactopyranodide) as a substrate
(Behringer, R. R., Crotty, D. A, Tennyson, V. A, Brinster, R. L.,
Palmiter, R. D. & Wolgemuth, D. J. (1993) Development 117:
823-833).
[0100] Preparation of Tissues for Immunostaining and Staining for
.beta.Galactosidase Activity--For immunohistochemical analyses,
mouse tissues were fixed in 4% paraformaldehyde and embedded in
paraffin. For analyses of .beta.-galactosidase expression, whole
embryos and postnatal tissues were fixed in 0.2% glutaraldehyde, 2%
paraformaldehyde in 0.1 M M phosphate buffer, pH 7.3, for 60 min at
4.degree. C. washed three times for 30 min with a 7.3 pH 0.1 M
phospate buffer containing 0.1% sodium deoxycholate, 2 mM MgCl2 and
then stained with X-Gal (1 mg/ml X-Gal, 5 mM K-ferricyanide, 5 mM
K-ferrocyanide) before embedding in paraffin. Experimental wounds
were made to transgenic mice by small cutaneous incisions, the
wounds were closed by a single suture, and the wounds surrounded by
normal skin were removed surgically after three and seven days and
processed for staining.
[0101] Immunohistochemical Staining--Five .mu.m thick paraffin
sections were stained with polyclonal antibodies against laminin-1
or the .gamma.2 chain of laminin-5. In brief the paraffin sections
were first incubated with 0.4% pepsin in 0.1 M HCl at 37.degree. C.
for 20 min to expose the antigens, blocked for nonspecific binding
with 5% newborn rabbit serum, 0.1% BSA, and then incubated for 1 at
37.degree. C. with the polygonal IgG diluted in TBS to 5-10
.mu.g/ml. Subsequently, a biotinylated swine-anti-rabbit antibody
was applied, followed by incubation with a 1:400 dilution of
Horseradish-Peroxidase-Avidin-Biotin-Complex (DAKO, Copenhagen,
Denmark). The color was developed in diaminobentsamidine (DAB),
followed by counterstaining of the slides with hematoxylin.
RESULTS
[0102] Characterization of Proteins and Epithelium-Derived
Cells--Immiunopurified trimeric laminin-5, isolated from the
culture medium of HaCat cells contained two major bands when
analyzed by SDS-PAGE (FIG. 1). These bands corresponded,
respectively, to the 165 kDa .alpha.3 chain, and the 155 kDa and
140 kDa .gamma.2 and .beta.3 chains migrating as a single band, as
reported previously (Carter, W. G., Ryan, M. C. & Gahr, P. J.
(1991) Cell 65: 599-610; Rousselle, P., Lunstrum, G. P., Keene, D.
R. & Burgeson, R. E. (1991) J. Cell Biol. 114: 567-576;
Pikkarainen, T., Schulthess, T., Engel, J. and Tryggvason, K.
(1992) Eur. J. Biochem. 209, 571-582). Additionally, a weak band of
about 105 kDa corresponding to the processed .gamma.2 chain could
be observed.
[0103] Full-length human recombinant laminin .gamma.2 chain was
produced in High-5 Spodoptera frugiperda insect cells using the
baculovirus system. Since the .gamma.2 chain was not secreted to
the culture medium, possibly because it was not assembled
intracellularly into a normal heterotrimer, it was isolated from
the cell fraction as described in Materials and Methods. The
protein was extracted under denaturating conditions using 5 M urea,
renatured by extensive dialysis against 50 mM Tris-HCl, 100 mM
NaCl, pH 7.4, and purified. The purified recombinant .gamma.2 chain
was full length and highly pure as determined by SDS-PAGE analysis
(FIG. 5).
[0104] The HaCat human keratinocytes and mouse KLN-205 squamous
carcinoma cells were shown to express laminin-5, based on northern
analyses and immunostaining, using a cDNA probe (Kallunki, P.,
Sainio, K., Eddy, R., Byers, M., Kallunki, T., Sariola, H., Beck,
K., Hirvonen, H., Shows, T. B. & Tryggvason, K. (1992) J. Cell
Biol. 119: 679-693) and/or polyclonal antibodies specific for the
.gamma.2 chain (Pyke, C., Salo, S., Ralfkiaer, E., Romer, J., Dano,
K. & Tryggvason, K. (1995) Cancer Res. 55: 4132-4139),
respectively. Furthermore, the KLN-205 cells developed .gamma.2
chain positive primary tumors and metasases in mice in vivo (data
not shown). Following intramuscular or subcutaneous inoculations,
large primary tumors developed in 4 weeks with numerous lung
metastases after 4-6 weeks. KLN-205 cells injected into the tail
vein produced multiple lung tumors (experimental metastases) in
four weeks. Consequently, both cell types were considered
appropriate for the cell attachment and migration experiments
carried out in this study.
[0105] Laminin-5 Molecule, but not Recombinant Laminin .gamma.2
Chair Promotes Cell Adhesion--The laminin-5 and recombinant
.gamma.2 chain prepared in this study, as well as commercial
laminin-1, were used as substrata in attachment assays (FIG. 6)
with the two epithelium-derived HaCat and KLN-205 cell lines that
both express laminin-5. Both cell lines attached about 2.5 times
more readily to laminin-1 than to plastic. Adhesion of the cells to
laminin-5 appeared to be slightly higher than that to laminin-1,
but the differences were not statistically significant. The cells
attached equally well to laminin-5 preparations denatured in 5 M
urea and then renatured by dialysis against 50 mM Tris-HCl, 100 mM
NaCl, pH 7.4, as described for the recombinant .gamma.2 chain
above, indicating that this treatment did not affect the binding
properties of the trimeric molecule. The attachment to laminin-5
was not significantly decreased in the presence of two different
polyclonal antibodies made against the short or long arms of the
.gamma.2 chain or pre-IgG. Different amounts of the antibody
against the short arm of the .gamma.2 chain were also tested (up to
50 .mu.g/ml), but no effects on cell adhesion were observed. When
the cells were plated on the recombinant .gamma.2 chain alone, the
attachment was not significantly higher than that to plastic, this
attachment not being influenced by polyclonal antibodies against
the .gamma.2 chain. The data confirm previous results showing that
trimeric laminin-5 promotes adhesion of epithelial cells, but the
present results further strongly suggest that this adhesion is not
mediated by the .gamma.2 chain.
[0106] Antibodies Against Laminin .gamma.2Domain III, but not
Domain I/II, Inhibit Cell Migration--Immunohistochemical and in
situ hybridization studies on healing skin wounds have indicated a
role for laminin-5 in cell migration. The potential role of the
.gamma.2 chain of laminin-5 in this process was examined for the
KLN-205 cells in vitro using Boyden and Transwell chamber assays as
described in Materials and Methods.
[0107] Migration was first studied in the Boyden chamber assay
using laminin-1 and fibronectin in the lower chamber as
chemoattractants. The cells were plated on type IV collagen coated
filters separating the upper and lower compartments, and antibodies
to different domains of the .gamma.2 chain were added to the upper
chamber. Migration of cells in the presence of preimmune IgG was
arbitrarily set as 100% (FIG. 7). When polyclonal IgG against the
short arm of the .gamma.2 chain was added to the upper compartment
containing the cells, the migration of cells through the filter was
decreased to about 35 to 45% of that observed with the preimmune
IgG (FIG. 7A). In contrast, the polyclonal IgG against C-terminal
domain I/II did not affect migration of the cells (FIG. 7A).
[0108] The effects of the two antibodies were similarly used in the
Transwell assay using native laminin-5 as chemoattractant in the
lower compartment, and the results were essentially the same as
above. Thus, addition of IgG raised against domain III of the
.gamma.2 chain inhibited the migration to about 50% as compared
with preimmune IgG, while the polyclonal IgG against domain I/II
did not affect the cell migration.
[0109] These in vitro results demonstrate that laminin-5 can have a
role in the locomotion of epithelium-derived cells, and that this
function can be inhibited by antibodies directed against domain III
of the .gamma.2 chain.
[0110] Limited Expression of LAMC2 Promoter-Reporter Gene
Constructs in Epithelial Cells in Transgenic Mice--In order to
search for potential epithelium-specific enhancer elements in the
LAMC2 gene, we made transgenic mice harboring DNA constructs
containing varying lengths of the 5' flanking region of the LAMC2
gene connected with a downstream reporter gene, which in this case
was LacZ coding for bacterial .beta.-galactosidase (SEQ. ID. NO.:
20)(FIG. 8). Two different constructs were used for microinjection
into pronuclei of fertilized mouse oozytes. The first construct
HH-1 contained an about 5,900 bp HindIII-SalI fragment, including
55 base pairs from the 5' untranslated region and the 5' flanking
region of the LAMC2 gene. The second construct, HH-2, contained 55
base pairs of the 5' untranslated region and 613 base pairs of the
5' flanking region. The sequence from the LAMC2 promoter region
cloned into HH-2 (FIG. 8) contains a GATAA box starting 27 base
pairs upstream of the transcription initiation site (Kallunki, P.,
Sainio, K., Eddy, R., Byers, M., Kallunki, T., Sariola, H., Beck,
K., Hirvonen, H., Shows, T. B. & Tryggvason, K. (1992) J. Cell
Biol. 119: 679-693), and two AP1 binding sites immediately upstream
Additionally, there are two Sp1 binding sides and an inverted CTF
sequence (CCAAT box) further upstream Three founder lines were
studied in detail for the expression pattern of HH-1, and two lines
for that of construct HH-2. Both constructs yielded similar
expression patterns for the .beta.-galactosidase reporter gene.
[0111] In mouse embryos very little expression was observed with
both constructs. In 15.5-day-old whole embryos only some hair
follicles, testicles and some regions of the skin showed positive
reaction (FIG. 9A). Microscopic analysis revealed positive stnig in
scattered basal keratinocytes of skin and some epithelial cells of
hair follicles and ductus deferens (FIG. 9B-D). Importantly, cells
of all other epithelia were negative for expression. These results
sharply contrast our previous results showing strong expression of
the LAMC2 gene in epithelial layers, including those of skin,
respiratory tract and kidney in human embryos as determined by in
situ hybridization (Kallunki, P., Sainio, K., Eddy, R., Byers, M.,
Kallunki, T., Sariola, H., Beck, K., Hirvonen, H., Shows, T. B.
& Tryggvason, K. (1992) J. Cell Biol. 119: 679-693). This shows
that the reporter gene constructs made in this study did not
contain the cis-acting elements necessary for epithelial
expression, although the restricted expression observed was limited
to only some epithelial cells.
[0112] Expression was also examined in tissues of adult transgenic
mice harboring the two constructs. Both constructs yielded highly
similar, but restricted expression in the embryos, whereas in the
adult tissues the distribution of expression was slightly more
extensive. Interestingly, as was the case for the embryos, the
limited expression was confined to epithelial cells, i.e. cell
types normally expressing only the LAMC2 gene in vivo. For example,
in skin discontinuous expression was observed in keratinocytes of
the epidermis and epithelial cells of some hair follicles (FIG.
10A). In the stomach intense expression could be seen in some villi
of the gastric mucosa, but it was absent in most areas (FIG.
10B,C). In positive areas expression could be seen in surface
epithelial cells and in cells of the gastric pits. As in the
embryos expression was also observed in epithelial cells of the
ductus deferens (FIG. 10D). However, epithelia such as those of the
respiratory tracts normally showing strong expression in vivo did
not show any expression at all in the present study.
[0113] Identification of a Cis-Element with Migration-Related
Activity in the LAMC2 Gene in Transgenic Mice--The in vitro cell
migration studies described above, together with previous
morphological studies have indicated a role for laminin-5 in cell
movement. These data imply that the genes for the subunits chains
of this protein should have regulatory elements induced during cell
migration. In order to further examine the association of cell
migration with laminin-S expression, we decided to initiate search
for potential migration-related cis-acting elements in the two
laminin .gamma.2 chain gene promoter-LacZ reporter gene constructs,
HH-1 and HH-2, using transgenic mice as a model system.
[0114] As we have previously shown, the LAMC2 gene is expressed in
keratinocytes migrating over a healing skin wound (Pyke, C., Romer,
J., Kallunki, P., Lund, L. R., Ralfkiaer, E., Dano, K. &
Tryggvason, K. (1994) Am. J. Pathol. 145: 782-791). According to
the present invention, small incision wounds were made into the
dorsal skin and tails of mice made transgenic with the two
constructs mentioned above to examine if the LacZ gene is expressed
in migrating keratinocytes during wound healing. As can be seen in
FIG. 11, immnunostaining of the dorsal skin wound showed distinct
linear staining for the laminin .gamma.2 chain in the normal
subepithelial basement membrane and also strong immunostaining for
this chain beneath keratinocytes migrating over the healing wound
could be seen. This confirms previous reports that laminin-5 is
present in the normal epithelial basement membrane and also that
the migrating keratinocytes express laminin-5. Staining with
polyclonal antibodies against EHS tumor laminin-1 containing the
.alpha.1, .beta.1 and .gamma.1 chains showed a weak staining of the
subepithelial basement membrane as well as staining of basement
membranes in blood vessels and muscle. Staining for expression of
the two .beta.-galactosidase reporter gene constructs showed
intense staining in keratinocytes migrating over the healing wound
(FIG. 11C,D), while no color reaction was noted in keratinocytes
resting on a normal epithelial basement membrane. The staining was
seen in several layers of keratinocytes indicating that the
enhancer element(s) directs expression in proliferating and
migrating keratinocytes. Identical patterns were obtained in mice
made with both constructs, demonstrating that the element necessary
for driving the expression is located within the 613 base pair
region immediately upstream of the transcription site of the LAMC2
gene.
[0115] Thus, according to the present invention, intact laminin-5
effectively mediates attachment of epithelial cells. As set forth
above in Example 4, the present invention utilized human
keratinocytes and KLN-205 mouse squamous carcinoma cells, both of
which were shown to express laminin-5. The effect of laminin-5 on
adhesion was similar to that of laminin-1 isolated from the mouse
EHS tumor. Both laminin isoforms have been shown to have similar
adhesive properties. This adhesion is presumably mediated through
.alpha.6.beta.4 and .alpha.3.beta.1 integrins that both bind to the
long arm of the laminin molecule. However, there is also an
indication that the short arm of the .gamma.2 chain is also
involved in the anchorage of epithelial cells, as an in-frame
deletion mutation removing 73 amino acid residues from domains III
and IV of the short arm of the .gamma.2 chain results in lethal
(Herlitz) junctional epidermolysis bullosa. In determining whether
this particular chain promoted cell adhesion adhesion studies were
carried out using full-length recombinant human .gamma.2 chain
produced in insect cells using the baculovirus system. It has been
shown by rotary shadowing that individually produced recombinant
.beta.1 and .gamma.1 chains maintain apparent normal tertiary
structure of the short arm (Pikkarainen, T., Schulthess, T., Engel,
J. and Tryggvason, K. (1992) Eur. J. Biochem. 209, 571-582), and,
therefore, the tertiary structure of the short arm of the .gamma.2
chain studied here was assumed to be normal and exposed. This
recombinant chain did not show any significant effects on cell
adhesion as compared with plastic. The recombinant chain was not
secreted to the cell culture medium, presumably because it was not
incorporated into a heterotrimeric molecule and, therefore, it
needed to be purified from the cytosol under denaturing conditions,
prior to renaturation. Although it is possible that this
polypeptide chain lost its adhesive properties due to the
denaturing conditions, it is not considered likely, as intact
laminin-5, also denatured and renatured using the same conditions,
maintained its adhesion activity. Furthermore, since it has been
shown that recombinant laminin a chains (or their domains) produced
in an eukaryotic expression system bind cellular receptors
(Yurchenco, P. D., U. Sung, M. Ward, Y. Yamada & J. J. O'Rear
(1993). J. Biol. Chem. 268: 8356-8365; Sung, U., J. J. O'Rear &
P. D. Yurchenco (1993). J. Cell Biol. 123: 1255-1268;
Colognato-Pyke, H., J. J. O'Rear, Y. Yamada, S. Carbonetto Y.-S.
Cheng, & P. D. Yurchenco (1995). J. Biol. Chem. 270: 9398-9406;
Colognato, H., M. MacCarrick, J. J. O'Rear & P. D. Yurchenco
(1997). J. Biol. Chem. 272: 29330-29336), it was concluded that the
recombinant .gamma.2 chain analyzed in the present study also
should be finctional. Thus, according to the present invention, the
short arm of the laminin .gamma.2 chain does not bind to cellular
receptors, and the important binding site lost in the above
mentioned lethal skin blistering disease interacts with some
protein(s) of the extracellular matrix This explanation is
plausible also because it has been shown that the E8 fragment of
laminin-1 containing the distal portion of the triple coiled coil
(long arm) and recombinant C-terminal G domain of the .alpha.1 and
.alpha.2 chains contains the cell binding sites (Yurchenco, P. D.,
U. Sung, M. Ward, Y. Yamada & J. J. O'Rear (1993). J. Biol.
Chem. 268: 8356-8365; Sung, U., J. J. O'Rear & P. D. Yurchenco
(1993). J. Cell Biol. 123: 1255-1268; Colognato-Pyke, H., J. J.
O'Rear, Y. Yamada, S. Carbonetto Y.-S. Cheng, & P. D. Yurchenco
(1995). J. Biol. Chem. 270: 9398-9406; Colognato, H., M.
MacCarrick, J. J. O'Rear & P. D. Yurchenco (1997). J. Biol.
Chem. 272: 29330-29336 Aumailley, M., Nurcombe, V., Edgar, D.,
Paulsson, M.N & Timpl, R. (1987) J. Biol. Chem. 262:
11532-11539). The laminin binding sites on the epithelial cells
have later been assigned to the integrins .alpha.6.beta.4 and
.alpha.3.beta.1. The conclusion that the short arm of the .gamma.2
chain does not interact with cellular receptors was further
supported by the present results showing that polyclonal antibodies
raised against the short arm do not inhibit adhesion of cells to
intact laminin-5.
[0116] Previous work has shown that expression of laminin-5 is
upregulated in migrating keratinocytes of healing wounds (Ryan, M.
C., Tizard, R., VanDevanter, D. R. & Carter, W. G. (1994) J.
Biol. Chem. 269: 22779-22787; Larjava, H., Salo, T., Haapasalmi K.,
Kramer, R. H. & Heino J. (1993) Clin. Invest. 92: 1425-1435;
Pyke, C., Romer, J., Kallunki, P., Lund, L. R., Ralfkiaer, E.,
Dano, K. & Tryggvason, K. (1994) Am. J. Pathol. 145: 782-791),
and also in tumor cells of invasive carcinomas (Pyke, C., Romer,
J., Kallunki, P., Lund, L. R., Ralfkiaer, E., Dano, K. &
Tryggvason, K. (1994) Am. J. Pathol. 145: 782-791; Pyke, C., Salo,
S., Ralfkiaer, E., Romer, J., Dano, K. & Tryggvason, K. (1995)
Cancer Res. 55: 4132-4139; Tani, T., Karttunen, T., Kiviluoto, T.,
Kivilaakso, E., Burgeson, R. E., Sipponen, P. & Virtanen, I.
(1996) Am. J. Pathol. 149: 781-793). Thus, this laminin isoform
could not only be surmised to be crucial for anchoring epithelial
cells to the underlying basement membrane but, additionally, a
protein of importance for anchoring epithelial cells or
epithelium-derived cancer cells to the extracellular environment
during cell migration. According to the results of Example 4 of the
present invention, antibodies against the short arm of the laminin
.gamma.2 chain inhibited the migration of KLN-205 squamous
carcinoma cells by about 55-65% as determined in the Boyden chamber
migration assay. Interestingly, the antibodies used here were
directed against 177 amino acid residues of domain III that when
deleted by mutation cause lethal junctional epidermolysis bullosa.
Accordingly, the short arm of the laminin .gamma.2 chain is
important for the interaction of this laminin isoform to other
extracellular matrix proteins, and this interaction is also crucial
for the migration process. Importantly, polyclonal antibodies
raised against the long arm of the .gamma.2 chain did not inhibit
migration of KLN-205 cells. Consequently, the results of the
present study demonstrate that the short arm of the .gamma.2 chain
is important for the adhesive function of laminin-5 similarly the
short arm of the laminin .gamma.1 chain that contains the only
known binding site of laminins for nidogen. This nidogen binding
site is essential for the formation of a bridge between the laminin
network on the one hand and the type IV collagen network and
perlecan on the other and, thus, it is necessary for the integrity
of the entire basement membrane matrix. The present and previous
data discussed above indicate that similarly to the .gamma.1 chain,
the laminin .gamma.12 chain possesses an important binding site
that anchors laminin-5 to some components of the extracellular
matrix. However, the laminin .gamma.2 chain does not bind to
nidogen .
[0117] The results of the experiments shown in Example 4 above with
the LAMC2 promoter-reporter gene constructs further emphasized the
involvement of the .gamma.2 chain and laminin-5 in cell migration,
as the reporter gene was strongly expressed in migratory cells of
healing wounds. The actual cis-acting element required for this
migration-related expression were not identified but it must be
located in the 613 base pair upstream region flanking the gene.
This region contains several motifs known to be important for gene
expression such as a GATAA box, AP-1 and Sp1 binding sites and a
CTF motif However, none of those have been shown to be associated
with cell migration. The results of the transgenic mouse
experiments carried out in this study further demonstrated that all
the enhancer elements necessary for driving normal expression in
epithelial cells are not present in a sequence reaching as far as
5,900 bp upstream of the transcription initiation site. Thus far,
no other tissue-specific enhancer elements have been reported for
any laminin gene.
[0118] Those skilled in the art will know, or be able to ascertain,
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. These and
all other equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
20 1 20 DNA Artificial Oligomer Primer 1 ggctcaccaa gacttacaca 20 2
20 DNA Artificial Oligomer Primer 2 gaatcactga gcagctgaac 20 3 20
DNA Artificial Oligomer Primer 3 cagtaccaga accgagttcg 20 4 20 DNA
Artificial Sequence Oligomer Primer 4 ctggttacca ggcttgagag 20 5 20
DNA Artificial Oligomer Primer 5 ttactgcgga atctcacagc 20 6 20 DNA
Artificial Oligomer Primer 6 tacactgttc aacccagggt 20 7 20 DNA
Artificial Oligomer Primer 7 aaacaagccc tctcactggt 20 8 20 DNA
Artificial Oligomer Primer 8 gcggagactg tgctgataag 20 9 20 DNA
Artificial Oligomer Primer 9 catacctctc tacatggcat 20 10 20 DNA
Artificial Oligomer Primer 10 agtctcgctg aatctctctt 20 11 20 DNA
Artificial Oligomer Primer 11 ttacaactag catggtgccc 20 12 5200 DNA
Homo sapiens sig_peptide (118)..(183) CDS (118)..(3699) polyA_site
(4433)..(4433) polyA_site (5195)..(5195) 12 gaccacctga tcgaaggaaa
aggaaggcac agcggagcgc agagtgagaa ccaccaaccg 60 aggcgccggg
cagcgacccc tgcagcggag acagagactg agcggcccgg caccgcc 117 atg cct gcg
ctc tgg ctg ggc tgc tgc ctc tgc ttc tcg ctc ctc ctg 165 Met Pro Ala
Leu Trp Leu Gly Cys Cys Leu Cys Phe Ser Leu Leu Leu 1 5 10 15 ccc
gca gcc cgg gcc acc tcc agg agg gaa gtc tgt gat tgc aat ggg 213 Pro
Ala Ala Arg Ala Thr Ser Arg Arg Glu Val Cys Asp Cys Asn Gly 20 25
30 aag tcc agg cag tgt atc ttt gat cgg gaa ctt cac aga caa act ggt
261 Lys Ser Arg Gln Cys Ile Phe Asp Arg Glu Leu His Arg Gln Thr Gly
35 40 45 aat gga ttc cgc tgc ctc aac tgc aat gac aac act gat ggc
att cac 309 Asn Gly Phe Arg Cys Leu Asn Cys Asn Asp Asn Thr Asp Gly
Ile His 50 55 60 tgc gag aag tgc aag aat ggc ttt tac cgg cac aga
gaa agg gac cgc 357 Cys Glu Lys Cys Lys Asn Gly Phe Tyr Arg His Arg
Glu Arg Asp Arg 65 70 75 80 tgt ttg ccc tgc aat tgt aac tcc aaa ggt
tct ctt agt gct cga tgt 405 Cys Leu Pro Cys Asn Cys Asn Ser Lys Gly
Ser Leu Ser Ala Arg Cys 85 90 95 gac aac tct gga cgg tgc agc tgt
aaa cca ggt gtg aca gga gcc aga 453 Asp Asn Ser Gly Arg Cys Ser Cys
Lys Pro Gly Val Thr Gly Ala Arg 100 105 110 tgc gac cga tgt ctg cca
ggc ttc cac atg ctc acg gat gcg ggg tgc 501 Cys Asp Arg Cys Leu Pro
Gly Phe His Met Leu Thr Asp Ala Gly Cys 115 120 125 acc caa gac cag
aga ctg cta gac tcc aag tgt gac tgt gac cca gct 549 Thr Gln Asp Gln
Arg Leu Leu Asp Ser Lys Cys Asp Cys Asp Pro Ala 130 135 140 ggc atc
gca ggg ccc tgt gac gcg ggc cgc tgt gtc tgc aag cca gct 597 Gly Ile
Ala Gly Pro Cys Asp Ala Gly Arg Cys Val Cys Lys Pro Ala 145 150 155
160 gtt act gga gaa cgc tgt gat agg tgt cga tca ggt tac tat aat ctg
645 Val Thr Gly Glu Arg Cys Asp Arg Cys Arg Ser Gly Tyr Tyr Asn Leu
165 170 175 gat ggg ggg aac cct gag ggc tgt acc cag tgt ttc tgc tat
ggg cat 693 Asp Gly Gly Asn Pro Glu Gly Cys Thr Gln Cys Phe Cys Tyr
Gly His 180 185 190 tca gcc agc tgc cgc agc tct gca gaa tac agt gtc
cat aag atc acc 741 Ser Ala Ser Cys Arg Ser Ser Ala Glu Tyr Ser Val
His Lys Ile Thr 195 200 205 tct acc ttt cat caa gat gtt gat ggc tgg
aag gct gtc caa cga aat 789 Ser Thr Phe His Gln Asp Val Asp Gly Trp
Lys Ala Val Gln Arg Asn 210 215 220 ggg tct cct gca aag ctc caa tgg
tca cag cgc cat caa gat gtg ttt 837 Gly Ser Pro Ala Lys Leu Gln Trp
Ser Gln Arg His Gln Asp Val Phe 225 230 235 240 agc tca gcc caa cga
cta gat cct gtc tat ttt gtg gct cct gcc aaa 885 Ser Ser Ala Gln Arg
Leu Asp Pro Val Tyr Phe Val Ala Pro Ala Lys 245 250 255 ttt ctt ggg
aat caa cag gtg agc tat ggg caa agc ctg tcc ttt gac 933 Phe Leu Gly
Asn Gln Gln Val Ser Tyr Gly Gln Ser Leu Ser Phe Asp 260 265 270 tac
cgt gtg gac aga gga ggc aga cac cca tct gcc cat gat gtg atc 981 Tyr
Arg Val Asp Arg Gly Gly Arg His Pro Ser Ala His Asp Val Ile 275 280
285 ctg gaa ggt gct ggt cta cgg atc aca gct ccc ttg atg cca ctt ggc
1029 Leu Glu Gly Ala Gly Leu Arg Ile Thr Ala Pro Leu Met Pro Leu
Gly 290 295 300 aag aca ctg cct tgt ggg ctc acc aag act tac aca ttc
agg tta aat 1077 Lys Thr Leu Pro Cys Gly Leu Thr Lys Thr Tyr Thr
Phe Arg Leu Asn 305 310 315 320 gag cat cca agc aat aat tgg agc ccc
cag ctg agt tac ttt gag tat 1125 Glu His Pro Ser Asn Asn Trp Ser
Pro Gln Leu Ser Tyr Phe Glu Tyr 325 330 335 cga agg tta ctg cgg aat
ctc aca gcc ctc cgc atc cga gct aca tat 1173 Arg Arg Leu Leu Arg
Asn Leu Thr Ala Leu Arg Ile Arg Ala Thr Tyr 340 345 350 gga gaa tac
agt act ggg tac att gac aat gtg acc ctg att tca gcc 1221 Gly Glu
Tyr Ser Thr Gly Tyr Ile Asp Asn Val Thr Leu Ile Ser Ala 355 360 365
cgc cct gtc tct gga gcc cca gca ccc tgg gtt gaa cag tgt ata tgt
1269 Arg Pro Val Ser Gly Ala Pro Ala Pro Trp Val Glu Gln Cys Ile
Cys 370 375 380 cct gtt ggg tac aag ggg caa ttc tgc cag gat tgt gct
tct ggc tac 1317 Pro Val Gly Tyr Lys Gly Gln Phe Cys Gln Asp Cys
Ala Ser Gly Tyr 385 390 395 400 aag aga gat tca gcg aga ctg ggg cct
ttt ggc acc tgt att cct tgt 1365 Lys Arg Asp Ser Ala Arg Leu Gly
Pro Phe Gly Thr Cys Ile Pro Cys 405 410 415 aac tgt caa ggg gga ggg
gcc tgt gat cca gac aca gga gat tgt tat 1413 Asn Cys Gln Gly Gly
Gly Ala Cys Asp Pro Asp Thr Gly Asp Cys Tyr 420 425 430 tca ggg gat
gag aat cct gac att gag tgt gct gac tgc cca att ggt 1461 Ser Gly
Asp Glu Asn Pro Asp Ile Glu Cys Ala Asp Cys Pro Ile Gly 435 440 445
ttc tac aac gat ccg cac gac ccc cgc agc tgc aag cca tgt ccc tgt
1509 Phe Tyr Asn Asp Pro His Asp Pro Arg Ser Cys Lys Pro Cys Pro
Cys 450 455 460 cat aac ggg ttc agc tgc tca gtg att ccg gag acg gag
gag gtg gtg 1557 His Asn Gly Phe Ser Cys Ser Val Ile Pro Glu Thr
Glu Glu Val Val 465 470 475 480 tgc aat aac tgc cct ccc ggg gtc acc
ggt gcc cgc tgt gag ctc tgt 1605 Cys Asn Asn Cys Pro Pro Gly Val
Thr Gly Ala Arg Cys Glu Leu Cys 485 490 495 gct gat ggc tac ttt ggg
gac ccc ttt ggt gaa cat ggc cca gtg agg 1653 Ala Asp Gly Tyr Phe
Gly Asp Pro Phe Gly Glu His Gly Pro Val Arg 500 505 510 cct tgt cag
ccc tgt caa tgc aac agc aat gtg gac ccc agt gcc tct 1701 Pro Cys
Gln Pro Cys Gln Cys Asn Ser Asn Val Asp Pro Ser Ala Ser 515 520 525
ggg aat tgt gac cgg ctg aca ggc agg tgt ttg aag tgt atc cac aac
1749 Gly Asn Cys Asp Arg Leu Thr Gly Arg Cys Leu Lys Cys Ile His
Asn 530 535 540 aca gcc ggc atc tac tgc gac cag tgc aaa gca ggc tac
ttc ggg gac 1797 Thr Ala Gly Ile Tyr Cys Asp Gln Cys Lys Ala Gly
Tyr Phe Gly Asp 545 550 555 560 cca ttg gct ccc aac cca gca gac aag
tgt cga gct tgc aac tgt aac 1845 Pro Leu Ala Pro Asn Pro Ala Asp
Lys Cys Arg Ala Cys Asn Cys Asn 565 570 575 ccc atg ggc tca gag cct
gta gga tgt cga agt gat ggc acc tgt gtt 1893 Pro Met Gly Ser Glu
Pro Val Gly Cys Arg Ser Asp Gly Thr Cys Val 580 585 590 tgc aag cca
gga ttt ggt ggc ccc aac tgt gag cat gga gca ttc agc 1941 Cys Lys
Pro Gly Phe Gly Gly Pro Asn Cys Glu His Gly Ala Phe Ser 595 600 605
tgt cca gct tgc tat aat caa gtg aag att cag atg gat cag ttt atg
1989 Cys Pro Ala Cys Tyr Asn Gln Val Lys Ile Gln Met Asp Gln Phe
Met 610 615 620 cag cag ctt cag aga atg gag gcc ctg att tca aag gct
cag ggt ggt 2037 Gln Gln Leu Gln Arg Met Glu Ala Leu Ile Ser Lys
Ala Gln Gly Gly 625 630 635 640 gat gga gta gta cct gat aca gag ctg
gaa ggc agg atg cag cag gct 2085 Asp Gly Val Val Pro Asp Thr Glu
Leu Glu Gly Arg Met Gln Gln Ala 645 650 655 gag cag gcc ctt cag gac
att ctg aga gat gcc cag att tca gaa ggt 2133 Glu Gln Ala Leu Gln
Asp Ile Leu Arg Asp Ala Gln Ile Ser Glu Gly 660 665 670 gct agc aga
tcc ctt ggt ctc cag ttg gcc aag gtg agg agc caa gag 2181 Ala Ser
Arg Ser Leu Gly Leu Gln Leu Ala Lys Val Arg Ser Gln Glu 675 680 685
aac agc tac cag agc cgc ctg gat gac ctc aag atg act gtg gaa aga
2229 Asn Ser Tyr Gln Ser Arg Leu Asp Asp Leu Lys Met Thr Val Glu
Arg 690 695 700 gtt cgg gct ctg gga agt cag tac cag aac cga gtt cgg
gat act cac 2277 Val Arg Ala Leu Gly Ser Gln Tyr Gln Asn Arg Val
Arg Asp Thr His 705 710 715 720 agg ctc atc act cag atg cag ctg agc
ctg gca gaa agt gaa gct tcc 2325 Arg Leu Ile Thr Gln Met Gln Leu
Ser Leu Ala Glu Ser Glu Ala Ser 725 730 735 ttg gga aac act aac att
cct gcc tca gac cac tac gtg ggg cca aat 2373 Leu Gly Asn Thr Asn
Ile Pro Ala Ser Asp His Tyr Val Gly Pro Asn 740 745 750 ggc ttt aaa
agt ctg gct cag gag gcc aca aga tta gca gaa agc cac 2421 Gly Phe
Lys Ser Leu Ala Gln Glu Ala Thr Arg Leu Ala Glu Ser His 755 760 765
gtt gag tca gcc agt aac atg gag caa ctg aca agg gaa act gag gac
2469 Val Glu Ser Ala Ser Asn Met Glu Gln Leu Thr Arg Glu Thr Glu
Asp 770 775 780 tat tcc aaa caa gcc ctc tca ctg gtg cgc aag gcc ctg
cat gaa gga 2517 Tyr Ser Lys Gln Ala Leu Ser Leu Val Arg Lys Ala
Leu His Glu Gly 785 790 795 800 gtc gga agc gga agc ggt agc ccg gac
ggt gct gtg gtg caa ggg ctt 2565 Val Gly Ser Gly Ser Gly Ser Pro
Asp Gly Ala Val Val Gln Gly Leu 805 810 815 gtg gaa aaa ttg gag aaa
acc aag tcc ctg gcc cag cag ttg aca agg 2613 Val Glu Lys Leu Glu
Lys Thr Lys Ser Leu Ala Gln Gln Leu Thr Arg 820 825 830 gag gcc act
caa gcg gaa att gaa gca gat agg tct tat cag cac agt 2661 Glu Ala
Thr Gln Ala Glu Ile Glu Ala Asp Arg Ser Tyr Gln His Ser 835 840 845
ctc cgc ctc ctg gat tca gtg tct ccg ctt cag gga gtc agt gat cag
2709 Leu Arg Leu Leu Asp Ser Val Ser Pro Leu Gln Gly Val Ser Asp
Gln 850 855 860 tcc ttt cag gtg gaa gaa gca aag agg atc aaa caa aaa
gcg gat tca 2757 Ser Phe Gln Val Glu Glu Ala Lys Arg Ile Lys Gln
Lys Ala Asp Ser 865 870 875 880 ctc tca agc ctg gta acc agg cat atg
gat gag ttc aag cgt aca caa 2805 Leu Ser Ser Leu Val Thr Arg His
Met Asp Glu Phe Lys Arg Thr Gln 885 890 895 aag aat ctg gga aac tgg
aaa gaa gaa gca cag cag ctc tta cag aat 2853 Lys Asn Leu Gly Asn
Trp Lys Glu Glu Ala Gln Gln Leu Leu Gln Asn 900 905 910 gga aaa agt
ggg aga gag aaa tca gat cag ctg ctt tcc cgt gcc aat 2901 Gly Lys
Ser Gly Arg Glu Lys Ser Asp Gln Leu Leu Ser Arg Ala Asn 915 920 925
ctt gct aaa agc aga gca caa gaa gca ctg agt atg ggc aat gcc act
2949 Leu Ala Lys Ser Arg Ala Gln Glu Ala Leu Ser Met Gly Asn Ala
Thr 930 935 940 ttt tat gaa gtt gag agc atc ctt aaa aac ctc aga gag
ttt gac ctg 2997 Phe Tyr Glu Val Glu Ser Ile Leu Lys Asn Leu Arg
Glu Phe Asp Leu 945 950 955 960 cag gtg gac aac aga aaa gca gaa gct
gaa gaa gcc atg aag aga ctc 3045 Gln Val Asp Asn Arg Lys Ala Glu
Ala Glu Glu Ala Met Lys Arg Leu 965 970 975 tcc tac atc agc cag aag
gtt tca gat gcc agt gac aag acc cag caa 3093 Ser Tyr Ile Ser Gln
Lys Val Ser Asp Ala Ser Asp Lys Thr Gln Gln 980 985 990 gca gaa aga
gcc ctg ggg agc gct gct gct gat gca cag agg gca aag 3141 Ala Glu
Arg Ala Leu Gly Ser Ala Ala Ala Asp Ala Gln Arg Ala Lys 995 1000
1005 aat ggg gcc ggg gag gcc ctg gaa atc tcc agt gag att gaa cag
3186 Asn Gly Ala Gly Glu Ala Leu Glu Ile Ser Ser Glu Ile Glu Gln
1010 1015 1020 gag att ggg agt ctg aac ttg gaa gcc aat gtg aca gca
gat gga 3231 Glu Ile Gly Ser Leu Asn Leu Glu Ala Asn Val Thr Ala
Asp Gly 1025 1030 1035 gcc ttg gcc atg gaa aag gga ctg gcc tct ctg
aag agt gag atg 3276 Ala Leu Ala Met Glu Lys Gly Leu Ala Ser Leu
Lys Ser Glu Met 1040 1045 1050 agg gaa gtg gaa gga gag ctg gaa agg
aag gag ctg gag ttt gac 3321 Arg Glu Val Glu Gly Glu Leu Glu Arg
Lys Glu Leu Glu Phe Asp 1055 1060 1065 acg aat atg gat gca gta cag
atg gtg att aca gaa gcc cag aag 3366 Thr Asn Met Asp Ala Val Gln
Met Val Ile Thr Glu Ala Gln Lys 1070 1075 1080 gtt gat acc aga gcc
aag aac gct ggg gtt aca atc caa gac aca 3411 Val Asp Thr Arg Ala
Lys Asn Ala Gly Val Thr Ile Gln Asp Thr 1085 1090 1095 ctc aac aca
tta gac ggc ctc ctg cat ctg atg gac cag cct ctc 3456 Leu Asn Thr
Leu Asp Gly Leu Leu His Leu Met Asp Gln Pro Leu 1100 1105 1110 agt
gta gat gaa gag ggg ctg gtc tta ctg gag cag aag ctt tcc 3501 Ser
Val Asp Glu Glu Gly Leu Val Leu Leu Glu Gln Lys Leu Ser 1115 1120
1125 cga gcc aag acc cag atc aac agc caa ctg cgg ccc atg atg tca
3546 Arg Ala Lys Thr Gln Ile Asn Ser Gln Leu Arg Pro Met Met Ser
1130 1135 1140 gag ctg gaa gag agg gca cgt cag cag agg ggc cac ctc
cat ttg 3591 Glu Leu Glu Glu Arg Ala Arg Gln Gln Arg Gly His Leu
His Leu 1145 1150 1155 ctg gag aca agc ata gat ggg att ctg gct gat
gtg aag aac ttg 3636 Leu Glu Thr Ser Ile Asp Gly Ile Leu Ala Asp
Val Lys Asn Leu 1160 1165 1170 gag aac att agg gac aac ctg ccc cca
ggc tgc tac aat acc cag 3681 Glu Asn Ile Arg Asp Asn Leu Pro Pro
Gly Cys Tyr Asn Thr Gln 1175 1180 1185 gct ctt gag caa cag tga
agctgccata aatatttctc aactgaggtt 3729 Ala Leu Glu Gln Gln 1190
cttgggatac agatctcagg gctcgggagc catgtcatgt gagtgggtgg gatggggaca
3789 tttgaacatg tttaatgggt atgctcaggt caactgacct gaccccattc
ctgatcccat 3849 ggccaggtgg ttgtcttatt gcaccatact ccttgcttcc
tgatgctggg catgaggcag 3909 ataggcactg gtgtgagaat gatcaaggat
ctggacccca aagatagact ggatggaaag 3969 acaaactgca caggcagatg
tttgcctcat aatagtcgta agtggagtcc tggaatttgg 4029 acaagtgctg
ttgggatata gtcaacttat tctttgagta atgtgactaa aggaaaaaac 4089
tttgactttg cccaggcatg aaattcttcc taatgtcaga acagagtgca acccagtcac
4149 actgtggcca gtaaaatact attgcctcat attgtcctct gcaagcttct
tgctgatcag 4209 agttcctcct acttacaacc cagggtgtga acatgttctc
cattttcaag ctggaagaag 4269 tgagcagtgt tggagtgagg acctgtaagg
caggcccatt cagagctatg gtgcttgctg 4329 gtgcctgcca ccttcaagtt
ctggacctgg gcatgacatc ctttctttta atgatgccat 4389 ggcaacttag
agattgcatt tttattaaag catttcctac cagcaaagca aatgttggga 4449
aagtatttac tttttcggtt tcaaagtgat agaaaagtgt ggcttgggca ttgaaagagg
4509 taaaattctc tagatttatt agtcctaatt caatcctact tttcgaacac
caaaaatgat 4569 gcgcatcaat gtattttatc ttattttctc aatctcctct
ctctttcctc cacccataat 4629 aagagaatgt tcctactcac acttcagctg
ggtcacatcc atccctccat tcatccttcc 4689 atccatcttt ccatccatta
cctccatcca tccttccaac atatatttat tgagtaccta 4749 ctgtgtgcca
ggggctggtg ggacagtggt gacatagtct ctgccctcat agagttgatt 4809
gtctagtgag gaagacaagc atttttaaaa aataaattta aacttacaaa ctttgtttgt
4869 cacaagtggt gtttattgca ataaccgctt ggtttgcaac ctctttgctc
aacagaacat 4929 atgttgcaag accctcccat gggcactgag tttggcaagg
atgacagagc tctgggttgt 4989 gcacatttct ttgcattcca gcgtcactct
gtgccttcta caactgattg caacagactg 5049 ttgagttatg ataacaccag
tgggaattgc tggaggaacc agaggcactt ccaccttggc 5109 tgggaagact
atggtgctgc cttgcttctg tatttccttg gattttcctg aaagtgtttt 5169
taaataaaga acaattgtta gatgccaaaa a 5200 13 1193 PRT Homo sapiens 13
Met Pro Ala Leu Trp Leu Gly Cys Cys Leu Cys Phe Ser Leu Leu Leu 1 5
10 15 Pro Ala Ala Arg Ala Thr Ser Arg Arg Glu Val Cys Asp Cys Asn
Gly 20 25 30 Lys Ser Arg Gln Cys Ile Phe Asp Arg Glu Leu His Arg
Gln Thr Gly 35 40 45 Asn Gly Phe Arg Cys Leu Asn Cys Asn Asp Asn
Thr Asp Gly Ile His 50
55 60 Cys Glu Lys Cys Lys Asn Gly Phe Tyr Arg His Arg Glu Arg Asp
Arg 65 70 75 80 Cys Leu Pro Cys Asn Cys Asn Ser Lys Gly Ser Leu Ser
Ala Arg Cys 85 90 95 Asp Asn Ser Gly Arg Cys Ser Cys Lys Pro Gly
Val Thr Gly Ala Arg 100 105 110 Cys Asp Arg Cys Leu Pro Gly Phe His
Met Leu Thr Asp Ala Gly Cys 115 120 125 Thr Gln Asp Gln Arg Leu Leu
Asp Ser Lys Cys Asp Cys Asp Pro Ala 130 135 140 Gly Ile Ala Gly Pro
Cys Asp Ala Gly Arg Cys Val Cys Lys Pro Ala 145 150 155 160 Val Thr
Gly Glu Arg Cys Asp Arg Cys Arg Ser Gly Tyr Tyr Asn Leu 165 170 175
Asp Gly Gly Asn Pro Glu Gly Cys Thr Gln Cys Phe Cys Tyr Gly His 180
185 190 Ser Ala Ser Cys Arg Ser Ser Ala Glu Tyr Ser Val His Lys Ile
Thr 195 200 205 Ser Thr Phe His Gln Asp Val Asp Gly Trp Lys Ala Val
Gln Arg Asn 210 215 220 Gly Ser Pro Ala Lys Leu Gln Trp Ser Gln Arg
His Gln Asp Val Phe 225 230 235 240 Ser Ser Ala Gln Arg Leu Asp Pro
Val Tyr Phe Val Ala Pro Ala Lys 245 250 255 Phe Leu Gly Asn Gln Gln
Val Ser Tyr Gly Gln Ser Leu Ser Phe Asp 260 265 270 Tyr Arg Val Asp
Arg Gly Gly Arg His Pro Ser Ala His Asp Val Ile 275 280 285 Leu Glu
Gly Ala Gly Leu Arg Ile Thr Ala Pro Leu Met Pro Leu Gly 290 295 300
Lys Thr Leu Pro Cys Gly Leu Thr Lys Thr Tyr Thr Phe Arg Leu Asn 305
310 315 320 Glu His Pro Ser Asn Asn Trp Ser Pro Gln Leu Ser Tyr Phe
Glu Tyr 325 330 335 Arg Arg Leu Leu Arg Asn Leu Thr Ala Leu Arg Ile
Arg Ala Thr Tyr 340 345 350 Gly Glu Tyr Ser Thr Gly Tyr Ile Asp Asn
Val Thr Leu Ile Ser Ala 355 360 365 Arg Pro Val Ser Gly Ala Pro Ala
Pro Trp Val Glu Gln Cys Ile Cys 370 375 380 Pro Val Gly Tyr Lys Gly
Gln Phe Cys Gln Asp Cys Ala Ser Gly Tyr 385 390 395 400 Lys Arg Asp
Ser Ala Arg Leu Gly Pro Phe Gly Thr Cys Ile Pro Cys 405 410 415 Asn
Cys Gln Gly Gly Gly Ala Cys Asp Pro Asp Thr Gly Asp Cys Tyr 420 425
430 Ser Gly Asp Glu Asn Pro Asp Ile Glu Cys Ala Asp Cys Pro Ile Gly
435 440 445 Phe Tyr Asn Asp Pro His Asp Pro Arg Ser Cys Lys Pro Cys
Pro Cys 450 455 460 His Asn Gly Phe Ser Cys Ser Val Ile Pro Glu Thr
Glu Glu Val Val 465 470 475 480 Cys Asn Asn Cys Pro Pro Gly Val Thr
Gly Ala Arg Cys Glu Leu Cys 485 490 495 Ala Asp Gly Tyr Phe Gly Asp
Pro Phe Gly Glu His Gly Pro Val Arg 500 505 510 Pro Cys Gln Pro Cys
Gln Cys Asn Ser Asn Val Asp Pro Ser Ala Ser 515 520 525 Gly Asn Cys
Asp Arg Leu Thr Gly Arg Cys Leu Lys Cys Ile His Asn 530 535 540 Thr
Ala Gly Ile Tyr Cys Asp Gln Cys Lys Ala Gly Tyr Phe Gly Asp 545 550
555 560 Pro Leu Ala Pro Asn Pro Ala Asp Lys Cys Arg Ala Cys Asn Cys
Asn 565 570 575 Pro Met Gly Ser Glu Pro Val Gly Cys Arg Ser Asp Gly
Thr Cys Val 580 585 590 Cys Lys Pro Gly Phe Gly Gly Pro Asn Cys Glu
His Gly Ala Phe Ser 595 600 605 Cys Pro Ala Cys Tyr Asn Gln Val Lys
Ile Gln Met Asp Gln Phe Met 610 615 620 Gln Gln Leu Gln Arg Met Glu
Ala Leu Ile Ser Lys Ala Gln Gly Gly 625 630 635 640 Asp Gly Val Val
Pro Asp Thr Glu Leu Glu Gly Arg Met Gln Gln Ala 645 650 655 Glu Gln
Ala Leu Gln Asp Ile Leu Arg Asp Ala Gln Ile Ser Glu Gly 660 665 670
Ala Ser Arg Ser Leu Gly Leu Gln Leu Ala Lys Val Arg Ser Gln Glu 675
680 685 Asn Ser Tyr Gln Ser Arg Leu Asp Asp Leu Lys Met Thr Val Glu
Arg 690 695 700 Val Arg Ala Leu Gly Ser Gln Tyr Gln Asn Arg Val Arg
Asp Thr His 705 710 715 720 Arg Leu Ile Thr Gln Met Gln Leu Ser Leu
Ala Glu Ser Glu Ala Ser 725 730 735 Leu Gly Asn Thr Asn Ile Pro Ala
Ser Asp His Tyr Val Gly Pro Asn 740 745 750 Gly Phe Lys Ser Leu Ala
Gln Glu Ala Thr Arg Leu Ala Glu Ser His 755 760 765 Val Glu Ser Ala
Ser Asn Met Glu Gln Leu Thr Arg Glu Thr Glu Asp 770 775 780 Tyr Ser
Lys Gln Ala Leu Ser Leu Val Arg Lys Ala Leu His Glu Gly 785 790 795
800 Val Gly Ser Gly Ser Gly Ser Pro Asp Gly Ala Val Val Gln Gly Leu
805 810 815 Val Glu Lys Leu Glu Lys Thr Lys Ser Leu Ala Gln Gln Leu
Thr Arg 820 825 830 Glu Ala Thr Gln Ala Glu Ile Glu Ala Asp Arg Ser
Tyr Gln His Ser 835 840 845 Leu Arg Leu Leu Asp Ser Val Ser Pro Leu
Gln Gly Val Ser Asp Gln 850 855 860 Ser Phe Gln Val Glu Glu Ala Lys
Arg Ile Lys Gln Lys Ala Asp Ser 865 870 875 880 Leu Ser Ser Leu Val
Thr Arg His Met Asp Glu Phe Lys Arg Thr Gln 885 890 895 Lys Asn Leu
Gly Asn Trp Lys Glu Glu Ala Gln Gln Leu Leu Gln Asn 900 905 910 Gly
Lys Ser Gly Arg Glu Lys Ser Asp Gln Leu Leu Ser Arg Ala Asn 915 920
925 Leu Ala Lys Ser Arg Ala Gln Glu Ala Leu Ser Met Gly Asn Ala Thr
930 935 940 Phe Tyr Glu Val Glu Ser Ile Leu Lys Asn Leu Arg Glu Phe
Asp Leu 945 950 955 960 Gln Val Asp Asn Arg Lys Ala Glu Ala Glu Glu
Ala Met Lys Arg Leu 965 970 975 Ser Tyr Ile Ser Gln Lys Val Ser Asp
Ala Ser Asp Lys Thr Gln Gln 980 985 990 Ala Glu Arg Ala Leu Gly Ser
Ala Ala Ala Asp Ala Gln Arg Ala Lys 995 1000 1005 Asn Gly Ala Gly
Glu Ala Leu Glu Ile Ser Ser Glu Ile Glu Gln 1010 1015 1020 Glu Ile
Gly Ser Leu Asn Leu Glu Ala Asn Val Thr Ala Asp Gly 1025 1030 1035
Ala Leu Ala Met Glu Lys Gly Leu Ala Ser Leu Lys Ser Glu Met 1040
1045 1050 Arg Glu Val Glu Gly Glu Leu Glu Arg Lys Glu Leu Glu Phe
Asp 1055 1060 1065 Thr Asn Met Asp Ala Val Gln Met Val Ile Thr Glu
Ala Gln Lys 1070 1075 1080 Val Asp Thr Arg Ala Lys Asn Ala Gly Val
Thr Ile Gln Asp Thr 1085 1090 1095 Leu Asn Thr Leu Asp Gly Leu Leu
His Leu Met Asp Gln Pro Leu 1100 1105 1110 Ser Val Asp Glu Glu Gly
Leu Val Leu Leu Glu Gln Lys Leu Ser 1115 1120 1125 Arg Ala Lys Thr
Gln Ile Asn Ser Gln Leu Arg Pro Met Met Ser 1130 1135 1140 Glu Leu
Glu Glu Arg Ala Arg Gln Gln Arg Gly His Leu His Leu 1145 1150 1155
Leu Glu Thr Ser Ile Asp Gly Ile Leu Ala Asp Val Lys Asn Leu 1160
1165 1170 Glu Asn Ile Arg Asp Asn Leu Pro Pro Gly Cys Tyr Asn Thr
Gln 1175 1180 1185 Ala Leu Glu Gln Gln 1190 14 4316 DNA Homo
sapiens sig_peptide (118)..(183) CDS (118)..(3453) repeat_unit
(4021)..(4316) repeat type is "other"; repeat family is "HUMAN
ALU"polyA_site (4296)..(4296) 14 gaccacctga tcgaaggaaa aggaaggcac
agcggagcgc agagtgagaa ccaccaaccg 60 aggcgccggg cagcgacccc
tgcagcggag acagagactg agcggcccgg caccgcc 117 atg cct gcg ctc tgg
ctg ggc tgc tgc ctc tgc ttc tcg ctc ctc ctg 165 Met Pro Ala Leu Trp
Leu Gly Cys Cys Leu Cys Phe Ser Leu Leu Leu 1 5 10 15 ccc gca gcc
cgg gcc acc tcc agg agg gaa gtc tgt gat tgc aat ggg 213 Pro Ala Ala
Arg Ala Thr Ser Arg Arg Glu Val Cys Asp Cys Asn Gly 20 25 30 aag
tcc agg cag tgt atc ttt gat cgg gaa ctt cac aga caa act ggt 261 Lys
Ser Arg Gln Cys Ile Phe Asp Arg Glu Leu His Arg Gln Thr Gly 35 40
45 aat gga ttc cgc tgc ctc aac tgc aat gac aac act gat ggc att cac
309 Asn Gly Phe Arg Cys Leu Asn Cys Asn Asp Asn Thr Asp Gly Ile His
50 55 60 tgc gag aag tgc aag aat ggc ttt tac cgg cac aga gaa agg
gac cgc 357 Cys Glu Lys Cys Lys Asn Gly Phe Tyr Arg His Arg Glu Arg
Asp Arg 65 70 75 80 tgt ttg ccc tgc aat tgt aac tcc aaa ggt tct ctt
agt gct cga tgt 405 Cys Leu Pro Cys Asn Cys Asn Ser Lys Gly Ser Leu
Ser Ala Arg Cys 85 90 95 gac aac tct gga cgg tgc agc tgt aaa cca
ggt gtg aca gga gcc aga 453 Asp Asn Ser Gly Arg Cys Ser Cys Lys Pro
Gly Val Thr Gly Ala Arg 100 105 110 tgc gac cga tgt ctg cca ggc ttc
cac atg ctc acg gat gcg ggg tgc 501 Cys Asp Arg Cys Leu Pro Gly Phe
His Met Leu Thr Asp Ala Gly Cys 115 120 125 acc caa gac cag aga ctg
cta gac tcc aag tgt gac tgt gac cca gct 549 Thr Gln Asp Gln Arg Leu
Leu Asp Ser Lys Cys Asp Cys Asp Pro Ala 130 135 140 ggc atc gca ggg
ccc tgt gac gcg ggc cgc tgt gtc tgc aag cca gct 597 Gly Ile Ala Gly
Pro Cys Asp Ala Gly Arg Cys Val Cys Lys Pro Ala 145 150 155 160 gtt
act gga gaa cgc tgt gat agg tgt cga tca ggt tac tat aat ctg 645 Val
Thr Gly Glu Arg Cys Asp Arg Cys Arg Ser Gly Tyr Tyr Asn Leu 165 170
175 gat ggg ggg aac cct gag ggc tgt acc cag tgt ttc tgc tat ggg cat
693 Asp Gly Gly Asn Pro Glu Gly Cys Thr Gln Cys Phe Cys Tyr Gly His
180 185 190 tca gcc agc tgc cgc agc tct gca gaa tac agt gtc cat aag
atc acc 741 Ser Ala Ser Cys Arg Ser Ser Ala Glu Tyr Ser Val His Lys
Ile Thr 195 200 205 tct acc ttt cat caa gat gtt gat ggc tgg aag gct
gtc caa cga aat 789 Ser Thr Phe His Gln Asp Val Asp Gly Trp Lys Ala
Val Gln Arg Asn 210 215 220 ggg tct cct gca aag ctc caa tgg tca cag
cgc cat caa gat gtg ttt 837 Gly Ser Pro Ala Lys Leu Gln Trp Ser Gln
Arg His Gln Asp Val Phe 225 230 235 240 agc tca gcc caa cga cta gat
cct gtc tat ttt gtg gct cct gcc aaa 885 Ser Ser Ala Gln Arg Leu Asp
Pro Val Tyr Phe Val Ala Pro Ala Lys 245 250 255 ttt ctt ggg aat caa
cag gtg agc tat ggg caa agc ctg tcc ttt gac 933 Phe Leu Gly Asn Gln
Gln Val Ser Tyr Gly Gln Ser Leu Ser Phe Asp 260 265 270 tac cgt gtg
gac aga gga ggc aga cac cca tct gcc cat gat gtg atc 981 Tyr Arg Val
Asp Arg Gly Gly Arg His Pro Ser Ala His Asp Val Ile 275 280 285 ctg
gaa ggt gct ggt cta cgg atc aca gct ccc ttg atg cca ctt ggc 1029
Leu Glu Gly Ala Gly Leu Arg Ile Thr Ala Pro Leu Met Pro Leu Gly 290
295 300 aag aca ctg cct tgt ggg ctc acc aag act tac aca ttc agg tta
aat 1077 Lys Thr Leu Pro Cys Gly Leu Thr Lys Thr Tyr Thr Phe Arg
Leu Asn 305 310 315 320 gag cat cca agc aat aat tgg agc ccc cag ctg
agt tac ttt gag tat 1125 Glu His Pro Ser Asn Asn Trp Ser Pro Gln
Leu Ser Tyr Phe Glu Tyr 325 330 335 cga agg tta ctg cgg aat ctc aca
gcc ctc cgc atc cga gct aca tat 1173 Arg Arg Leu Leu Arg Asn Leu
Thr Ala Leu Arg Ile Arg Ala Thr Tyr 340 345 350 gga gaa tac agt act
ggg tac att gac aat gtg acc ctg att tca gcc 1221 Gly Glu Tyr Ser
Thr Gly Tyr Ile Asp Asn Val Thr Leu Ile Ser Ala 355 360 365 cgc cct
gtc tct gga gcc cca gca ccc tgg gtt gaa cag tgt ata tgt 1269 Arg
Pro Val Ser Gly Ala Pro Ala Pro Trp Val Glu Gln Cys Ile Cys 370 375
380 cct gtt ggg tac aag ggg caa ttc tgc cag gat tgt gct tct ggc tac
1317 Pro Val Gly Tyr Lys Gly Gln Phe Cys Gln Asp Cys Ala Ser Gly
Tyr 385 390 395 400 aag aga gat tca gcg aga ctg ggg cct ttt ggc acc
tgt att cct tgt 1365 Lys Arg Asp Ser Ala Arg Leu Gly Pro Phe Gly
Thr Cys Ile Pro Cys 405 410 415 aac tgt caa ggg gga ggg gcc tgt gat
cca gac aca gga gat tgt tat 1413 Asn Cys Gln Gly Gly Gly Ala Cys
Asp Pro Asp Thr Gly Asp Cys Tyr 420 425 430 tca ggg gat gag aat cct
gac att gag tgt gct gac tgc cca att ggt 1461 Ser Gly Asp Glu Asn
Pro Asp Ile Glu Cys Ala Asp Cys Pro Ile Gly 435 440 445 ttc tac aac
gat ccg cac gac ccc cgc agc tgc aag cca tgt ccc tgt 1509 Phe Tyr
Asn Asp Pro His Asp Pro Arg Ser Cys Lys Pro Cys Pro Cys 450 455 460
cat aac ggg ttc agc tgc tca gtg att ccg gag acg gag gag gtg gtg
1557 His Asn Gly Phe Ser Cys Ser Val Ile Pro Glu Thr Glu Glu Val
Val 465 470 475 480 tgc aat aac tgc cct ccc ggg gtc acc ggt gcc cgc
tgt gag ctc tgt 1605 Cys Asn Asn Cys Pro Pro Gly Val Thr Gly Ala
Arg Cys Glu Leu Cys 485 490 495 gct gat ggc tac ttt ggg gac ccc ttt
ggt gaa cat ggc cca gtg agg 1653 Ala Asp Gly Tyr Phe Gly Asp Pro
Phe Gly Glu His Gly Pro Val Arg 500 505 510 cct tgt cag ccc tgt caa
tgc aac agc aat gtg gac ccc agt gcc tct 1701 Pro Cys Gln Pro Cys
Gln Cys Asn Ser Asn Val Asp Pro Ser Ala Ser 515 520 525 ggg aat tgt
gac cgg ctg aca ggc agg tgt ttg aag tgt atc cac aac 1749 Gly Asn
Cys Asp Arg Leu Thr Gly Arg Cys Leu Lys Cys Ile His Asn 530 535 540
aca gcc ggc atc tac tgc gac cag tgc aaa gca ggc tac ttc ggg gac
1797 Thr Ala Gly Ile Tyr Cys Asp Gln Cys Lys Ala Gly Tyr Phe Gly
Asp 545 550 555 560 cca ttg gct ccc aac cca gca gac aag tgt cga gct
tgc aac tgt aac 1845 Pro Leu Ala Pro Asn Pro Ala Asp Lys Cys Arg
Ala Cys Asn Cys Asn 565 570 575 ccc atg ggc tca gag cct gta gga tgt
cga agt gat ggc acc tgt gtt 1893 Pro Met Gly Ser Glu Pro Val Gly
Cys Arg Ser Asp Gly Thr Cys Val 580 585 590 tgc aag cca gga ttt ggt
ggc ccc aac tgt gag cat gga gca ttc agc 1941 Cys Lys Pro Gly Phe
Gly Gly Pro Asn Cys Glu His Gly Ala Phe Ser 595 600 605 tgt cca gct
tgc tat aat caa gtg aag att cag atg gat cag ttt atg 1989 Cys Pro
Ala Cys Tyr Asn Gln Val Lys Ile Gln Met Asp Gln Phe Met 610 615 620
cag cag ctt cag aga atg gag gcc ctg att tca aag gct cag ggt ggt
2037 Gln Gln Leu Gln Arg Met Glu Ala Leu Ile Ser Lys Ala Gln Gly
Gly 625 630 635 640 gat gga gta gta cct gat aca gag ctg gaa ggc agg
atg cag cag gct 2085 Asp Gly Val Val Pro Asp Thr Glu Leu Glu Gly
Arg Met Gln Gln Ala 645 650 655 gag cag gcc ctt cag gac att ctg aga
gat gcc cag att tca gaa ggt 2133 Glu Gln Ala Leu Gln Asp Ile Leu
Arg Asp Ala Gln Ile Ser Glu Gly 660 665 670 gct agc aga tcc ctt ggt
ctc cag ttg gcc aag gtg agg agc caa gag 2181 Ala Ser Arg Ser Leu
Gly Leu Gln Leu Ala Lys Val Arg Ser Gln Glu 675 680 685 aac agc tac
cag agc cgc ctg gat gac ctc aag atg act gtg gaa aga 2229 Asn Ser
Tyr Gln Ser Arg Leu Asp Asp Leu Lys Met Thr Val Glu Arg 690 695 700
gtt cgg gct ctg gga agt cag tac cag aac cga gtt cgg gat act cac
2277 Val Arg Ala Leu Gly Ser Gln Tyr Gln Asn Arg Val Arg Asp Thr
His 705 710 715 720 agg ctc atc act cag atg cag ctg agc ctg gca gaa
agt gaa gct tcc 2325 Arg Leu Ile Thr Gln Met Gln Leu Ser Leu Ala
Glu Ser Glu Ala Ser 725 730 735 ttg gga aac act aac att cct gcc tca
gac cac tac gtg ggg cca aat 2373 Leu Gly Asn Thr Asn Ile Pro Ala
Ser Asp His Tyr Val Gly Pro Asn 740 745 750 ggc ttt aaa agt ctg gct
cag gag gcc aca aga tta gca gaa agc cac 2421 Gly Phe Lys Ser Leu
Ala Gln Glu Ala Thr Arg Leu Ala Glu Ser His 755 760 765 gtt gag tca
gcc agt aac atg gag caa ctg aca agg gaa act gag gac 2469 Val Glu
Ser Ala Ser Asn Met Glu Gln Leu Thr Arg Glu Thr Glu Asp 770 775 780
tat tcc aaa caa gcc ctc tca ctg gtg cgc aag gcc ctg cat gaa gga
2517 Tyr Ser Lys Gln Ala Leu Ser Leu Val Arg Lys Ala Leu His Glu
Gly 785 790 795 800 gtc gga agc gga agc ggt agc ccg gac ggt gct gtg
gtg caa ggg ctt 2565 Val Gly Ser Gly Ser Gly Ser Pro Asp Gly Ala
Val Val Gln Gly Leu 805 810 815 gtg gaa aaa ttg gag aaa acc aag tcc
ctg gcc cag cag ttg aca agg 2613 Val Glu Lys Leu Glu Lys Thr Lys
Ser Leu Ala Gln Gln Leu Thr Arg 820 825 830 gag gcc act caa gcg gaa
att gaa gca gat agg tct tat cag cac agt 2661 Glu Ala Thr Gln Ala
Glu Ile Glu Ala Asp Arg Ser Tyr Gln His Ser 835 840 845 ctc cgc ctc
ctg gat tca gtg tct ccg ctt cag gga gtc agt gat cag 2709 Leu Arg
Leu Leu Asp Ser Val Ser Pro Leu Gln Gly Val Ser Asp Gln 850 855 860
tcc ttt cag gtg gaa gaa gca aag agg atc aaa caa aaa gcg gat tca
2757 Ser Phe Gln Val Glu Glu Ala Lys Arg Ile Lys Gln Lys Ala Asp
Ser 865 870 875 880 ctc tca agc ctg gta acc agg cat atg gat gag ttc
aag cgt aca caa 2805 Leu Ser Ser Leu Val Thr Arg His Met Asp Glu
Phe Lys Arg Thr Gln 885 890 895 aag aat ctg gga aac tgg aaa gaa gaa
gca cag cag ctc tta cag aat 2853 Lys Asn Leu Gly Asn Trp Lys Glu
Glu Ala Gln Gln Leu Leu Gln Asn 900 905 910 gga aaa agt ggg aga gag
aaa tca gat cag ctg ctt tcc cgt gcc aat 2901 Gly Lys Ser Gly Arg
Glu Lys Ser Asp Gln Leu Leu Ser Arg Ala Asn 915 920 925 ctt gct aaa
agc aga gca caa gaa gca ctg agt atg ggc aat gcc act 2949 Leu Ala
Lys Ser Arg Ala Gln Glu Ala Leu Ser Met Gly Asn Ala Thr 930 935 940
ttt tat gaa gtt gag agc atc ctt aaa aac ctc aga gag ttt gac ctg
2997 Phe Tyr Glu Val Glu Ser Ile Leu Lys Asn Leu Arg Glu Phe Asp
Leu 945 950 955 960 cag gtg gac aac aga aaa gca gaa gct gaa gaa gcc
atg aag aga ctc 3045 Gln Val Asp Asn Arg Lys Ala Glu Ala Glu Glu
Ala Met Lys Arg Leu 965 970 975 tcc tac atc agc cag aag gtt tca gat
gcc agt gac aag acc cag caa 3093 Ser Tyr Ile Ser Gln Lys Val Ser
Asp Ala Ser Asp Lys Thr Gln Gln 980 985 990 gca gaa aga gcc ctg ggg
agc gct gct gct gat gca cag agg gca aag 3141 Ala Glu Arg Ala Leu
Gly Ser Ala Ala Ala Asp Ala Gln Arg Ala Lys 995 1000 1005 aat ggg
gcc ggg gag gcc ctg gaa atc tcc agt gag att gaa cag 3186 Asn Gly
Ala Gly Glu Ala Leu Glu Ile Ser Ser Glu Ile Glu Gln 1010 1015 1020
gag att ggg agt ctg aac ttg gaa gcc aat gtg aca gca gat gga 3231
Glu Ile Gly Ser Leu Asn Leu Glu Ala Asn Val Thr Ala Asp Gly 1025
1030 1035 gcc ttg gcc atg gaa aag gga ctg gcc tct ctg aag agt gag
atg 3276 Ala Leu Ala Met Glu Lys Gly Leu Ala Ser Leu Lys Ser Glu
Met 1040 1045 1050 agg gaa gtg gaa gga gag ctg gaa agg aag gag ctg
gag ttt gac 3321 Arg Glu Val Glu Gly Glu Leu Glu Arg Lys Glu Leu
Glu Phe Asp 1055 1060 1065 acg aat atg gat gca gta cag atg gtg att
aca gaa gcc cag aag 3366 Thr Asn Met Asp Ala Val Gln Met Val Ile
Thr Glu Ala Gln Lys 1070 1075 1080 gtt gat acc aga gcc aag aac gct
ggg gtt aca atc caa gac aca 3411 Val Asp Thr Arg Ala Lys Asn Ala
Gly Val Thr Ile Gln Asp Thr 1085 1090 1095 ctc aac aca tta gac ggc
ctc ctg cat ctg atg ggt atg tga 3453 Leu Asn Thr Leu Asp Gly Leu
Leu His Leu Met Gly Met 1100 1105 1110 acccacaacc cacaaccttc
cagctccatg ctccagggct ttgctccaga acactcacta 3513 tacctagccc
cagcaaaggg gagtctcagc tttccttaag gatatcagta aatgtgcttt 3573
gtttccaggc ccagataact ttcggcaggt tcccttacat ttactggacc ctgttttacc
3633 gttgctaaga tgggtcactg aacacctatt gcacttgggg gtaaaggtct
gtgggccaaa 3693 gaacaggtgt atataagcaa cttcacagaa cacgagacag
cttgggaatc ctgctaaaga 3753 gtctggcctg gaccctgaga agccagtgga
cagttttaag cagaggaata acatcaccac 3813 tgtatatttc agaaagatca
ctagggcagc cgagtggagg aaagcttgaa gagggggtta 3873 gagagaaggc
aggttgagac tacttaagat attgttgaaa taattgaaga gagaaatgac 3933
aggagcctgc tctaaggcag tagaatggtg gctgggaaga tgtgaaggaa gattttccca
3993 gtctgtgaag tcaagaatca cttgccggcc gggtgtggtg gctcacgcct
gtaattctag 4053 cactttggga gactgaagcg ggtggatcac ccgaggtcag
gagttgaaga ccagcctggc 4113 caacatggtg aaaccctgtc tctactaaaa
gtacaaaaat tagctggatg atggtggtgg 4173 gcgcctgtaa ttccagctac
tcaggagtct gaggcaggag aatcgcttga acccaggagg 4233 cgaggttaca
gtgagccaag attgcaccac tgctcttcca gcctgggaac agagagactg 4293
cctaaaaaaa aaaaaaaaaa aaa 4316 15 1111 PRT Homo sapiens 15 Met Pro
Ala Leu Trp Leu Gly Cys Cys Leu Cys Phe Ser Leu Leu Leu 1 5 10 15
Pro Ala Ala Arg Ala Thr Ser Arg Arg Glu Val Cys Asp Cys Asn Gly 20
25 30 Lys Ser Arg Gln Cys Ile Phe Asp Arg Glu Leu His Arg Gln Thr
Gly 35 40 45 Asn Gly Phe Arg Cys Leu Asn Cys Asn Asp Asn Thr Asp
Gly Ile His 50 55 60 Cys Glu Lys Cys Lys Asn Gly Phe Tyr Arg His
Arg Glu Arg Asp Arg 65 70 75 80 Cys Leu Pro Cys Asn Cys Asn Ser Lys
Gly Ser Leu Ser Ala Arg Cys 85 90 95 Asp Asn Ser Gly Arg Cys Ser
Cys Lys Pro Gly Val Thr Gly Ala Arg 100 105 110 Cys Asp Arg Cys Leu
Pro Gly Phe His Met Leu Thr Asp Ala Gly Cys 115 120 125 Thr Gln Asp
Gln Arg Leu Leu Asp Ser Lys Cys Asp Cys Asp Pro Ala 130 135 140 Gly
Ile Ala Gly Pro Cys Asp Ala Gly Arg Cys Val Cys Lys Pro Ala 145 150
155 160 Val Thr Gly Glu Arg Cys Asp Arg Cys Arg Ser Gly Tyr Tyr Asn
Leu 165 170 175 Asp Gly Gly Asn Pro Glu Gly Cys Thr Gln Cys Phe Cys
Tyr Gly His 180 185 190 Ser Ala Ser Cys Arg Ser Ser Ala Glu Tyr Ser
Val His Lys Ile Thr 195 200 205 Ser Thr Phe His Gln Asp Val Asp Gly
Trp Lys Ala Val Gln Arg Asn 210 215 220 Gly Ser Pro Ala Lys Leu Gln
Trp Ser Gln Arg His Gln Asp Val Phe 225 230 235 240 Ser Ser Ala Gln
Arg Leu Asp Pro Val Tyr Phe Val Ala Pro Ala Lys 245 250 255 Phe Leu
Gly Asn Gln Gln Val Ser Tyr Gly Gln Ser Leu Ser Phe Asp 260 265 270
Tyr Arg Val Asp Arg Gly Gly Arg His Pro Ser Ala His Asp Val Ile 275
280 285 Leu Glu Gly Ala Gly Leu Arg Ile Thr Ala Pro Leu Met Pro Leu
Gly 290 295 300 Lys Thr Leu Pro Cys Gly Leu Thr Lys Thr Tyr Thr Phe
Arg Leu Asn 305 310 315 320 Glu His Pro Ser Asn Asn Trp Ser Pro Gln
Leu Ser Tyr Phe Glu Tyr 325 330 335 Arg Arg Leu Leu Arg Asn Leu Thr
Ala Leu Arg Ile Arg Ala Thr Tyr 340 345 350 Gly Glu Tyr Ser Thr Gly
Tyr Ile Asp Asn Val Thr Leu Ile Ser Ala 355 360 365 Arg Pro Val Ser
Gly Ala Pro Ala Pro Trp Val Glu Gln Cys Ile Cys 370 375 380 Pro Val
Gly Tyr Lys Gly Gln Phe Cys Gln Asp Cys Ala Ser Gly Tyr 385 390 395
400 Lys Arg Asp Ser Ala Arg Leu Gly Pro Phe Gly Thr Cys Ile Pro Cys
405 410 415 Asn Cys Gln Gly Gly Gly Ala Cys Asp Pro Asp Thr Gly Asp
Cys Tyr 420 425 430 Ser Gly Asp Glu Asn Pro Asp Ile Glu Cys Ala Asp
Cys Pro Ile Gly 435 440 445 Phe Tyr Asn Asp Pro His Asp Pro Arg Ser
Cys Lys Pro Cys Pro Cys 450 455 460 His Asn Gly Phe Ser Cys Ser Val
Ile Pro Glu Thr Glu Glu Val Val 465 470 475 480 Cys Asn Asn Cys Pro
Pro Gly Val Thr Gly Ala Arg Cys Glu Leu Cys 485 490 495 Ala Asp Gly
Tyr Phe Gly Asp Pro Phe Gly Glu His Gly Pro Val Arg 500 505 510 Pro
Cys Gln Pro Cys Gln Cys Asn Ser Asn Val Asp Pro Ser Ala Ser 515 520
525 Gly Asn Cys Asp Arg Leu Thr Gly Arg Cys Leu Lys Cys Ile His Asn
530 535 540 Thr Ala Gly Ile Tyr Cys Asp Gln Cys Lys Ala Gly Tyr Phe
Gly Asp 545 550 555 560 Pro Leu Ala Pro Asn Pro Ala Asp Lys Cys Arg
Ala Cys Asn Cys Asn 565 570 575 Pro Met Gly Ser Glu Pro Val Gly Cys
Arg Ser Asp Gly Thr Cys Val 580 585 590 Cys Lys Pro Gly Phe Gly Gly
Pro Asn Cys Glu His Gly Ala Phe Ser 595 600 605 Cys Pro Ala Cys Tyr
Asn Gln Val Lys Ile Gln Met Asp Gln Phe Met 610 615 620 Gln Gln Leu
Gln Arg Met Glu Ala Leu Ile Ser Lys Ala Gln Gly Gly 625 630 635 640
Asp Gly Val Val Pro Asp Thr Glu Leu Glu Gly Arg Met Gln Gln Ala 645
650 655 Glu Gln Ala Leu Gln Asp Ile Leu Arg Asp Ala Gln Ile Ser Glu
Gly 660 665 670 Ala Ser Arg Ser Leu Gly Leu Gln Leu Ala Lys Val Arg
Ser Gln Glu 675 680 685 Asn Ser Tyr Gln Ser Arg Leu Asp Asp Leu Lys
Met Thr Val Glu Arg 690 695 700 Val Arg Ala Leu Gly Ser Gln Tyr Gln
Asn Arg Val Arg Asp Thr His 705 710 715 720 Arg Leu Ile Thr Gln Met
Gln Leu Ser Leu Ala Glu Ser Glu Ala Ser 725 730 735 Leu Gly Asn Thr
Asn Ile Pro Ala Ser Asp His Tyr Val Gly Pro Asn 740 745 750 Gly Phe
Lys Ser Leu Ala Gln Glu Ala Thr Arg Leu Ala Glu Ser His 755 760 765
Val Glu Ser Ala Ser Asn Met Glu Gln Leu Thr Arg Glu Thr Glu Asp 770
775 780 Tyr Ser Lys Gln Ala Leu Ser Leu Val Arg Lys Ala Leu His Glu
Gly 785 790 795 800 Val Gly Ser Gly Ser Gly Ser Pro Asp Gly Ala Val
Val Gln Gly Leu 805 810 815 Val Glu Lys Leu Glu Lys Thr Lys Ser Leu
Ala Gln Gln Leu Thr Arg 820 825 830 Glu Ala Thr Gln Ala Glu Ile Glu
Ala Asp Arg Ser Tyr Gln His Ser 835 840 845 Leu Arg Leu Leu Asp Ser
Val Ser Pro Leu Gln Gly Val Ser Asp Gln 850 855 860 Ser Phe Gln Val
Glu Glu Ala Lys Arg Ile Lys Gln Lys Ala Asp Ser 865 870 875 880 Leu
Ser Ser Leu Val Thr Arg His Met Asp Glu Phe Lys Arg Thr Gln 885 890
895 Lys Asn Leu Gly Asn Trp Lys Glu Glu Ala Gln Gln Leu Leu Gln Asn
900 905 910 Gly Lys Ser Gly Arg Glu Lys Ser Asp Gln Leu Leu Ser Arg
Ala Asn 915 920 925 Leu Ala Lys Ser Arg Ala Gln Glu Ala Leu Ser Met
Gly Asn Ala Thr 930 935 940 Phe Tyr Glu Val Glu Ser Ile Leu Lys Asn
Leu Arg Glu Phe Asp Leu 945 950 955 960 Gln Val Asp Asn Arg Lys Ala
Glu Ala Glu Glu Ala Met Lys Arg Leu 965 970 975 Ser Tyr Ile Ser Gln
Lys Val Ser Asp Ala Ser Asp Lys Thr Gln Gln 980 985 990 Ala Glu Arg
Ala Leu Gly Ser Ala Ala Ala Asp Ala Gln Arg Ala Lys 995 1000 1005
Asn Gly Ala Gly Glu Ala Leu Glu Ile Ser Ser Glu Ile Glu Gln 1010
1015 1020 Glu Ile Gly Ser Leu Asn Leu Glu Ala Asn Val Thr Ala Asp
Gly 1025 1030 1035 Ala Leu Ala Met Glu Lys Gly Leu Ala Ser Leu Lys
Ser Glu Met 1040 1045 1050 Arg Glu Val Glu Gly Glu Leu Glu Arg Lys
Glu Leu Glu Phe Asp 1055 1060 1065 Thr Asn Met Asp Ala Val Gln Met
Val Ile Thr Glu Ala Gln Lys 1070 1075 1080 Val Asp Thr Arg Ala Lys
Asn Ala Gly Val Thr Ile Gln Asp Thr 1085 1090 1095 Leu Asn Thr Leu
Asp Gly Leu Leu His Leu Met Gly Met 1100 1105 1110 16 20 DNA
Artificial Oligomer Primer 16 gagcgcagag tgagaaccac 20 17 20 DNA
Artificial Oligomer Primer 17 actgtattct gcagagctgc 20 18 20 DNA
Artificial Oligomer Primer 18 ttcctttccc ctaccttgtg 20 19 20 DNA
Artificial Oligomer Primer 19 tgtggaagcc tggcagacat 20 20 720 PRT
Homo sapiens 20 Ala Gly Thr Cys Thr Thr Thr Ala Thr Ala Gly Gly Gly
Ala Gly Gly 1 5 10 15 Thr Thr Gly Gly Cys Cys Ala Gly Thr Cys Ala
Ala Thr Ala Gly Gly 20 25 30 Thr Thr Ala Cys Thr Thr Thr Ala Thr
Gly Ala Gly Thr Thr Gly Cys 35 40 45 Thr Ala Ala Cys Cys Cys Thr
Gly Gly Thr Gly Ala Gly Cys Ala Gly 50 55 60 Gly Ala Ala Gly Thr
Thr Ala Thr Gly Thr Gly Gly Ala Cys Cys Ala 65 70 75 80 Gly Gly Ala
Gly Ala Gly Ala Ala Ala Cys Cys Cys Thr Thr Gly Gly 85 90 95 Thr
Thr Cys Ala Gly Cys Cys Thr Gly Gly Ala Gly Ala Ala Ala Gly 100 105
110 Gly Ala Gly Ala Gly Gly Thr Thr Gly Ala Cys Cys Cys Thr Ala Ala
115 120 125 Ala Cys Thr Gly Gly Ala Gly Gly Gly Thr Gly Gly Ala Gly
Ala Gly 130 135 140 Gly Ala Cys Cys Cys Thr Gly Thr Thr Gly Thr Gly
Ala Cys Thr Cys 145 150 155 160 Thr Cys Cys Gly Ala Cys Thr Gly Ala
Cys Thr Thr Gly Thr Cys Thr 165 170 175 Thr Cys Cys Thr Thr Gly Ala
Thr Gly Thr Cys Cys Thr Thr Thr Ala 180 185 190 Ala Gly Cys Cys Gly
Gly Ala Gly Cys Thr Gly Ala Thr Thr Cys Gly 195 200 205 Gly Gly Cys
Thr Gly Cys Thr Gly Cys Cys Thr Thr Ala Thr Thr Thr 210 215 220 Cys
Thr Gly Ala Gly Thr Thr Ala Gly Cys Gly Cys Thr Cys Thr Thr 225 230
235 240 Ala Ala Gly Ala Thr Thr Gly Gly Gly Cys Cys Thr Cys Cys Cys
Ala 245 250 255 Gly Thr Thr Thr Gly Ala Gly Gly Ala Ala Gly Gly Gly
Gly Cys Gly 260 265 270 Gly Gly Cys Thr Gly Cys Thr Gly Thr Cys Thr
Ala Cys Cys Thr Cys 275 280 285 Thr Gly Thr Gly Ala Ala Thr Cys Thr
Gly Cys Cys Cys Thr Gly Gly 290 295 300 Ala Cys Cys Ala Cys Cys Cys
Cys Gly Gly Gly Ala Gly Ala Gly Ala 305 310 315 320 Ala Gly Gly Ala
Gly Gly Gly Cys Thr Cys Cys Gly Gly Gly Gly Ala 325 330 335 Ala Thr
Cys Thr Cys Gly Cys Ala Cys Ala Thr Thr Cys Cys Ala Gly 340 345 350
Gly Cys Ala Ala Ala Gly Gly Cys Thr Cys Cys Cys Gly Gly Gly Cys 355
360 365 Cys Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Thr Gly Cys Cys
Ala 370 375 380 Cys Ala Cys Cys Cys Thr Thr Gly Gly Cys Cys Cys Gly
Gly Gly Cys 385 390 395 400 Cys Ala Gly Gly Thr Gly Thr Gly Cys Gly
Cys Cys Cys Thr Cys Cys 405 410 415 Thr Cys Gly Cys Thr Gly Cys Gly
Ala Gly Gly Gly Gly Gly Ala Gly 420 425 430 Cys Gly Gly Gly Cys Gly
Gly Cys Thr Gly Cys Gly Gly Gly Gly Ala 435 440 445 Gly Cys Gly Ala
Thr Thr Thr Thr Cys Cys Ala Gly Cys Cys Cys Gly 450 455 460 Gly Thr
Thr Thr Gly Thr Gly Cys Thr Cys Thr Gly Thr Gly Thr Gly 465 470 475
480 Thr Thr Thr Gly Thr Cys Thr Gly Cys Cys Thr Cys Thr Gly Gly Ala
485 490 495 Gly Gly Gly Cys Thr Gly Gly Gly Thr Cys Cys Thr Cys Cys
Thr Thr 500 505 510 Ala Thr Thr Cys Ala Cys Ala Gly Gly Thr Gly Ala
Gly Thr Cys Ala 515 520 525 Cys Ala Cys Cys Cys Thr Gly Ala Ala Ala
Cys Ala Cys Ala Gly Gly 530 535 540 Cys Thr Cys Thr Cys Thr Thr Cys
Cys Thr Gly Thr Cys Ala Gly Gly 545 550 555 560 Ala Cys Thr Gly Ala
Gly Thr Cys Ala Gly Gly Thr Ala Gly Ala Ala 565 570 575 Gly Ala Gly
Thr Cys Gly Ala Thr Ala Ala Ala Ala Cys Cys Ala Cys 580 585 590
Cys Thr Gly Ala Thr Cys Ala Ala Gly Gly Ala Ala Ala Ala Gly Gly 595
600 605 Ala Ala Gly Gly Cys Ala Cys Ala Gly Cys Gly Gly Ala Gly Cys
Gly 610 615 620 Cys Ala Gly Ala Gly Thr Gly Ala Gly Ala Ala Cys Cys
Ala Cys Cys 625 630 635 640 Ala Ala Cys Cys Gly Ala Gly Gly Cys Gly
Cys Cys Gly Gly Gly Cys 645 650 655 Ala Gly Cys Gly Ala Cys Cys Cys
Cys Thr Gly Cys Ala Gly Cys Gly 660 665 670 Gly Ala Gly Ala Cys Ala
Gly Ala Gly Ala Cys Thr Gly Ala Gly Cys 675 680 685 Gly Gly Cys Cys
Cys Gly Gly Cys Ala Cys Cys Gly Cys Cys Ala Thr 690 695 700 Gly Cys
Cys Thr Gly Cys Gly Cys Thr Cys Thr Gly Gly Cys Thr Gly 705 710 715
720
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