U.S. patent application number 10/227738 was filed with the patent office on 2003-05-29 for laminin chains: diagnostic and therapeutic use.
This patent application is currently assigned to BioStratum AB. Invention is credited to Kallunki, Pekka, Pyke, Charles, Tryggvason, Karl.
Application Number | 20030100529 10/227738 |
Document ID | / |
Family ID | 23233705 |
Filed Date | 2003-05-29 |
United States Patent
Application |
20030100529 |
Kind Code |
A1 |
Tryggvason, Karl ; et
al. |
May 29, 2003 |
Laminin chains: diagnostic and therapeutic use
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; (La Jolla, CA) ; Pyke,
Charles; (Hilleroo, DK) |
Correspondence
Address: |
Richard J. Minnich
FAY, SHARPE, FAGAN, MINNICH & McKEE, LLP
Seventh Floor
1100 Superior Avenue
Cleveland
OH
44114-2518
US
|
Assignee: |
BioStratum AB
|
Family ID: |
23233705 |
Appl. No.: |
10/227738 |
Filed: |
August 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10227738 |
Aug 26, 2002 |
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09663147 |
Sep 15, 2000 |
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09663147 |
Sep 15, 2000 |
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08800593 |
Feb 18, 1997 |
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6143505 |
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08800593 |
Feb 18, 1997 |
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08317450 |
Oct 4, 1994 |
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5660982 |
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Current U.S.
Class: |
514/44R ; 435/5;
435/6.16; 435/6.17 |
Current CPC
Class: |
C12Q 1/6886 20130101;
G01N 33/57419 20130101; G01N 2474/20 20210801; G01N 33/5743
20130101; G01N 33/57484 20130101; C07K 16/28 20130101; Y10S 436/813
20130101; G01N 2333/78 20130101; C12Q 2600/158 20130101; G01N
33/57411 20130101 |
Class at
Publication: |
514/44 ; 435/5;
435/6 |
International
Class: |
A61K 048/00; C12Q
001/70; C12Q 001/68 |
Claims
We claim:
1. A method for detecting kalinin/laminin 5 expression in cells and
tissue comprising detecting a signal from the tissue assayed, such
signal resulting from specifically hybridizing the tissue with an
effective amount of a nucleic acid probe, which probe contains a
sense or antisense portion of the kalinin/laminin 5 gamma-2 chain
nucleic acid sequence.
2. The method of claim 1 where the nucleic acid probe is DNA.
3. The method of claim 1 where the nucleic acid probe is RNA.
4. The method of claim 1 where the nucleic acid probe is
radiolabelled, enzyme labelled, chemiluminescent labelled, avidin
or biotin labelled.
5. The method of claim 1 where the nucleic acid probe derived from
human kalinin/laminin 5 gamma-2 chain nucleic acid sequence.
6. The method of claim 1 where the nucleic acid probe is
incorporated into an extrachromasomal self-replicating vector.
7. The method of claim 1 where the nucleic acid probe is
incorporated into a viral vector.
8. The method of claim 1 where the nucleic acid probe is
linear.
9. The method of claim 1 where the nucleic acid probe is
circularized.
10. The method of claim 1 where the nucleic acid probe contains
modified nucleotides.
11. A method for detecting the presence of invasive cells in tissue
comprising detecting a signal from the tissue assayed, such signal
resulting from specifically hybridizing the tissue with an
effective amount of a nucleic acid probe, which probe contains a
sense or antisense portion of kalinin/laminin 5 gamma-2 chain
nucleic acid sequence.
12. The method of claim 11 where the nucleic acid probe is DNA.
13. The method of claim 11 where the nucleic acid probe is RNA.
14. The method of claim 11 where the nucleic acid probe is
radiolabelled, enzyme labelled, chemiluminescent labelled, avidin
or biotin labelled.
15. The method of claim 11 where the nucleic acid probe derived
from human kalinin/laminin 5 gamma-2 chain nucleic acid
sequence.
16. The method of claim 11 where the nucleic acid probe is
incorporated into an extrachromasomal self-replicating vector.
17. The method of claim 11 where the nucleic acid probe is
incorporated into a viral vector.
18. The method of claim 11 where the nucleic acid probe is
linear.
19. The method of claim 11 where the nucleic acid probe is
circularized.
20. The method of claim 11 where the nucleic acid probe contains
modified nucleotides.
21. A method for monitoring the presence of invasive cells in
tissue comprising detecting a signal or absence of signal from the
tissue assayed, such signal resulting from specifically hybridizing
the tissue with an effective amount of a nucleic acid probe, which
probe contains a sense or antisense portion of kalinin/laminin 5
gamma-2 chain nucleic acid sequence.
22. The method of claim 21 where the nucleic acid probe is DNA.
23. The method of claim 21 where the nucleic acid probe is RNA.
24. The method of claim 21 where the nucleic acid probe is
radiolabelled, enzyme labelled, cheruluminescent labelled, avidin
or biotin labelled.
25. The method of claim 21 where the nucleic acid probe derived
from human kalinin/laminin 5 gamma-2 chain nucleic acid
sequence.
26. The method of claim 21 where the nucleic acid probe is
incorporated into an extrachromasomal self-replicating vector.
27. The method of claim 21 where the nucleic acid probe is
incorporated into a viral vector.
28. The method of claim 21 where the nucleic acid probe is
linear.
29. The method of claim 21 where the nucleic acid probe is
circularized.
30. The method of claim 21 where the nucleic acid probe contains
modified nucleotides.
31. A method for detecting kalinin/laminin 5 expression in cells
and 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 gamma-2 chain protein.
32. 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 gamma-2 chain protein.
33. A method for monitoring invasive cells in malignant tissue
comprising detecting a signal from assayed malignant 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 gamma-2 chain protein.
Description
BACKGROUND OF THE INVENTION
[0001] 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 .alpha. chain
(.about.400 kd) and two light chains, .beta. and .gamma. (130-200
kd) (nomenclature according to Burgeson et al., Matrix Biol., 1994,
14:209-211). Laminin exists in numerous isoforms that are formed by
different combinations of laminin chain varients which currently
amount to at least nine.
[0002] Kalinin/laminin 5 (most likely also identical to the
adhesion molecule nicein) is a recently identified laminin isoform
which is a functional adhesion component for epithelial cells
(Tryggvason, 1993, supra.; Burgeson et al., 1994, supra.; Rousselle
et al., J. Cell Bio., 1991, 114:567-576; Kallunki et al., J. Cell
Biol., 1992, 119:679-693; Marinkovich et al., J. Biol. Chem., 1992,
267:17900-17906; Vailly et al., Eur. J. Biochem., 1994,
219:209-218). Kalinin/laminin 5 contains unique laminin varient
chains, one of which, the .gamma.2 chain, has recently been cloned
and sequenced (Kallunki et al., 1992, supra., previously named
B2t). The .gamma.2 chain has a mass of .about.130 kd and is thus
smaller than the "classical" .about.200 kd .beta.1 and .gamma.1
light chains of laminin. The domain structure of the .gamma.2 chain
also differs from that of the .gamma.1 chain in that it lacks the
amino-terminal globular domain (domain VI) believed to function in
intermolecular cross-linking of laminin molecules to form networks
(Yurcheno and O'Rear, in Molecular and Cellular Aspects of Basement
Membranes, 1993, (ed. Rohrbach and Timpl, Academic Press, San
Diego, pp. 20-47). In addition, domains III, IV, and V (containing
EGF-like repeats) in .gamma.2 are shorter than in the .gamma.1
chain (Kallunki et al., 1992, supra.).
[0003] 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).
[0004] 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 B 1 (.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 memebrane receptors, many of which are members of the integin
family (Ruoslati, E. J Clin. Invest. 1991, 87:1-5; Mecham, R. P.
FASEB J., 1991, 5:2538-2546; Hynes, R. Cell, 1992, 69:11-25).
[0005] Recently a new nomenclature for describing laminin has been
agreed to as in the following Table 1 (after Burgeson et al., 1994,
supra.)
1TABLE 1 laminin chains and genes heterotrimers of laminin New
Previous Gene New Chains Previous .alpha.1 A, Ae LAMA1 laminin-1
.alpha.1.beta.1.gamma.1 EHS laminin .alpha.2 M, Am LAMA2 laminin-2
.alpha.2.beta.1.gamma.1 merosin .alpha.3 200 kDa LAMA3 laminin-3
.alpha.1.beta.2.gamma.1 s-laminin .beta.1 B1, B1e LAMB1 laminin-4
.alpha.2.beta.2.gamma.1 s-merosin .beta.2 S, B1s LAMB2 laminin-5
.alpha.3.beta.3.gamma.2 kalinin/nicein .beta.3 140 kDa LAMB3
laminin-6 .alpha.3.beta.1.gamma.1 k-laminin .gamma.1 B2, B2e LAMC1
laminin-7 .alpha.3.beta.2.gamma.1 ks-laminin .gamma.2 B2t LAMC2
SUMMARY OF THE INVENTION
[0006] 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, radiolabelled, enzyme labelled, chemiluminescent labelled,
avidin or biotin labelled, 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 contiains modified nucleotides. In the
preferred embodiment the probes are linearized specific regions of
the .gamma.2 gene.
[0007] 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, radiolabelled, enzyme labelled, chemiluminescent labelled,
avidin or biotin labelled, 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.
[0008] 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
kalinin/laminin 5 gamma-2 protein.
[0009] 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 gamma-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 portion 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] 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 pbb2r-02.
Magnification:1A.times.100; 1B-1D.times.640.
[0011] 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). Magnification: 2C.times.100,
all others .times.640.
[0012] FIG. 3 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.
[0013] FIG. 4 shows the nucelic 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/GenBan/DDBJ under the accession number Z15008. FIG. 4B is the
nucleotide and derived amino acid sequence of the alternative 3'
end sequence from cDNA clones providing a sequence of 4,316 base
pairs, available from EMB/GenBank/DDBJ under the accession number
Z15009. (Kallunki et al., 1992, supra.).
DETAILED DESCRIPTION OF THE INVENTION
[0014] 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 protien
kalinin/nicein/epiligrin 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 specimins.
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.
[0015] Since the majority of cases are of the generalized (Herlitz)
phenotype (H-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. Longtern survival is rare.
[0016] 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.
[0017] 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.
[0018] 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 tumor
budding and subsequent prognosis.
[0019] 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.
[0020] 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 determine the level of .gamma.2 chain
expression as a measure of budding activity of a given tumor.
[0021] 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.
[0022] 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 nucelic 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.
[0023] 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.
[0024] 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 homologous recombination into
genomic DNA, targeted transposon insertion, or random
insertion/deletion mutations in the genomic DNA.
[0025] 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).
[0026] Thus the instant invention provides for a method of
detection, diagnosis, prognosis, monitoring, and therapeutic
treatment of invasive cell phenotypes.
[0027] The examples below are meant by way of illustration, and are
not meant to be limiting as to the scope of the instant
disclosure.
EXAMPLE 1
Mutation in the .gamma.2 Chain (Gene LAMC2 is Critical in Some
Cases of JFB
[0028] 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.
[0029] A Point Mutation Produces Exon Skipping
[0030] When a panel of five unrelated JEB patients were analysed,
the primers used to amplify segment C (nt 1046-1537) produced
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
nuclotide sequences within this and other PCR products did not
reveal any additional mutations upon MDE analysis.
[0031] 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).
[0032] 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,
predicting a truncated kalinin/laminin 5 .gamma.2 chain terminating
at residue 830.
[0033] RT-PCR and MDE Analyses
[0034] 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.
[0035] For PCR amplification, 1 .mu.L of cDNA was used as template
and amplification conditions were 94 C. for 5 min followed by 95 C.
for 45 sec, 60 C. for 45 sec and 72 C for 45 see 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
reccomendation (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.
[0036] 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.500 ng of genomic DNA was used as template and the
following oligomer primers were utilized.
2 5' GGCTCACCAAGACTTACACA 3'; 5' GAATCACTGAGCAGCTGAAC 3'; 5'
CAGTACCAGAACCGAGTTCG 3'; 5' CTGGTTACCAGGCTTGAGAG 3'; 5'
TTACTGCGGAATCTCACAGC 3'; 5' TACACTGTTCAACCCAGGGT 3'; 5'
AAACAAGCCCTCTCACTGGT 3'; 5' GCGGAGACTGTGCTGATAAG 3'; 5'
CATACCTCTCTACATGGCAT 3'; 5' AGTCTCGCTGAATCTCTCTT 3'; 5'
TTACAACTAGCATGGTGCCC 3'.
[0037] Amplification conditions were 94 C. for 7 min followed by 95
C. for 1.5 min, 56 C. (intron 8) or 58 C. (intron 16) for 1 min and
72 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
(Boehtringer Mannheim). The PCR products were subcloned and
sequenced as above.
[0038] Verification of Mutations
[0039] 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.
[0040] Amplification conditions were 94 C. for 7 min followed by 94
C. for 1 min, 58 C. for 45 sec and 72 C. for 45 sec for 35 cycles
in an OmniGene thermal cycler (Marsh Scientific). PCR products were
analysed on 2.5% agarose gels.
[0041] 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 exon skipping, 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 .gamma.2 Chain Gene LAMC2 is Critical in H-JEB
[0042] 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 occurrence of a homozygous premature tennination codon mutation
is the specific cause in an examined case of H-JEB (Aberdam et al.,
Nature Genetics, 1994, 6:299-304).
[0043] 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.
[0044] 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.
[0045] No major differences were detected in the 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 (FIG.
5a). 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.
[0046] 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 clevage 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.
[0047] Cell Culture
[0048] Epidermis was separated from dermis by dispase treatment at
37 C. keratinocytes were dissociated in 0.25% trypsin at 37 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-glutarine, 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.
[0049] Northern Blot Analysis
[0050] 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 Hyperfilm MP (Amersham) with intensifying screens. Radiolabeled
cDNA probes NA1 (Baudoin et al., J. Invest. Dermatol., 1994, in
press), KAL-5.5C (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.
[0051] RT-PCR and Heteroduplex Analysis (MDE)
[0052] 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', (R) 5'-ACTGTATTCTGCAGAGCTGC-3'. PCR
cycling conditions were: 94 C., 5 min, followed by 94 C., 45 sec;
60 C., 45 sec; 72 C., 45 sec; for 35 cycles, and extension at 72 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.
[0053] Vefication of the Mutation
[0054] PCR reactions on genomic DNA (50 .mu.g) were carried out
using the upstream primer 5'-TTCCTTTCCCCTACCTTGTG-3' and the
downstream primer 5'-TGTGGAAGCCTGGCAGACAT-3', which are located in
the intron 2 and exon 3 of LAMC2 respectively. PCR conditions were:
95 C., 5 min, followed by 94 C., 45 sec; 56 C., 45 sec; 72 C., 45
sec; for 35 cycles, and extension at 72 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). Clevage products were electrophoresed (2.4%
agarose) stained and visualized under UV light.
[0055] 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
.gamma.2 chain in forming proper cellular contacts.
EXAMPLE 3
.gamma.2 Chain as Diagnostic for Invasive Tissues
[0056] 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., October 1994, 145(4): 1-10 in
press).
[0057] 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
malingnant fibrous histiocytomas, 1 neurofibrosarcoma).
[0058] All samples were selected upon histological examination of a
hematoxylin and eosin-stained 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 bv Romer et
al. (J. Invest. Dermatol., 1994, 102:519-522), was fixed and
paraffin-embedded the same way as the human cancer specimens.
[0059] 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, in press).
[0060] Probes
[0061] Fragments of the cDNA for the .gamma.2 chain of human
kalinin/laminin 5 was 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 L
15 .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 EMBU/GenBank/DDBJ database as reported by Kallunki et al.,
(1992, supra.; FIG. 4).
[0062] 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 phb1e-01 (3460-4366
(accession No. J02778, Pikkarainen et al., J. Biol. Chem., 1987,
262:10454-10462) in pKS(Bluescript)II(+)); chain .gamma.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)).
[0063] 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 (Dyr) to an average size of 100 bases. RNA probes
transcribed from opposite strands of the same plasmid template,
yielding sense and anti-sense transcripts were adjusted to
1.times.10.sup.6 cpm/.mu.L and stored at -20 C. until used. Probes
were applied to tissue sections.
[0064] In Situ Hybridization
[0065] In situ Hybridization was performed as described by Pyke et
al., (Am. J. Pathol., 1991, 38:1059-1067) with S.sup.35 labeled RNA
probes prepared as described above. In brief, paraffin sections
were cut, placed on gelatinized slides, heated to 60 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 .mu.g/.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 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 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 C. for 15 minutes. After treatment with RNAse A (20 .mu.g/mL)
in NTE at 37 C. for 30 minutes, the sections were washed in NTE at
37 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 air dried. Finally, autoradiographic emulsion was
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.
[0066] Results: Laminin .alpha.1. .beta.1. .gamma.1. and .gamma.2
Chains
[0067] All rounds of in situr 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.
[0068] Colon Adenocarcinoma
[0069] 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.
[0070] 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.
[0071] Weak signals for laminin chains all, .gamma.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 samples
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.
[0072] 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 pbb2r-02. 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.
[0073] Ductal Mammara 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., .beta.1, and
.gamma.1 mRNAs were detected in fibroblast-like stromal cells
throughout cancerous areas in one of the cases.
[0076] Malignant Melanoma
[0077] 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.
[0078] Squamous Cell Carcinomas
[0079] 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).
[0080] 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 occured 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).
[0081] 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 indivtdual
malignant glandular structures are negative for .gamma.2 chain mRNA
(straight arrows). Note the wedge shaped form of the invading
glandular tissue. (AR 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 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 patter 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.
[0082] Sarcomas
[0083] All six sarcomas tested in the study were totally negative
for .gamma.2 chain mRNA. The expression of other laminin chains was
not done.
[0084] Mouse Wounded Skin
[0085] 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 epidermal tongue moving under the wound clot (FIG. 3). In
adjacent normal-looking skin, keratinocytes were negative for
.gamma.2 chain mRNA.
[0086] FIG. 3 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). 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 2A. Magnification:
.times.640.
[0087] RNAse Protection Assay
[0088] Plasmid phbt-03 was linearized with EcoRI and a radiolabeled
RNA-anti-sense probe was prepared by transcription using P-32 UTP
and T3 polymerase (Pyke et al., FEBS Letters, 1993, 326:69-75).
RNAse protection assay, using 40 .mu.g ethanol-precipitated and
DNAse 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.
[0089] 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.
[0090] 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.
[0091] 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.
* * * * *