Vol. 2, 447-456,
March
Clinical
1996
Expression
of CD44
Variant
Isoforms
Simone
Seiter,
Dirk Schadendorf,
Karin Herrmann,
Markus
Schneider,
Marc
R#{246}sel,Robert
and
Margot
Department
69120
Arch,
Wolfgang
Tilgen,
Z#{246}1ler
and Immune
Neuenheimer
University
[S. S., W. T.],
Defense,
Feld
German
280,
Cancer
69120
Hospital,
Department
Hospitalstrasse
of Tumor
Research
Heidelberg
3,
Center,
a variety
of the
described
of
human
adhesion
[S. S., K. H., M.
with
on the expression
melanomas,
and
primary
tumors,
molecule
as correlating
report
Exon
vlO is present
expression
expression
CD44
tumor
in thick
Thirteen
nevi,
39 cutaneous
(CD44v)
progression.
nocytes
and
a panel
surface
expression
variant
exons
melanoma
of the
standard
v5, v6, v7, v7-v8,
any
CD44
splice
been
Here,
and
we
as primary
a varying
Exons
specimens.
tissue
vlO was
metastases.
CD44v5-positive
flu/flu
mouse.
Because
was
forms,
node
metastases
rather
was
excluded.
one
might
tumors
frequently,
form,
lines
sion
of
distinct
as well
by expression
between
malignant
of CD44
of CD44
or tumor
expression
transformation
Considering
speculate
the function
that
expression
growth
of
of
=
(n
the
of melanovariant
CD44
Furthermore,
tumor
iso-
of human
studies,
colon,
expression
mammary,
and some
only
of
30,
33).
mating
with
other
with
from
even
tumor
In
not correlate
isoforms,
cervix
with
a still
variant
the
and
revised
10/4/95;
accepted
1 1/20/95.
‘ This
investigation
was
supported
by the Tumorzentrum
Heidelberg/
Mannheim
(M. Z., W. T.), the Mildred Scheelstiftung
f#{252}r
Krebshilfe
(M. Z., D. S.), the Sandozstiftung
f#{252}r
Therapeutische
Forschung
(M. Z.),
and Deutsche
Forschungsgemeinschaft
Grant Zo40-5/l
(M. Z.).
2 To whom
requests for reprints should be addressed,
at Department
of
Tumor Progression
and Immune
Defense,
German
Cancer Research
Center,
Im Neuenheimer
Feld 280,
6221-422454;
Fax: 6221-424760.
69120
Heidelberg,
Germany.
Phone:
sion
did
melanomas
increasing
3
(17),
It was
of published
provided
(18-34).
However,
carcinoma,
and
cell
expression
of CD44v
and/or
seem
from
squamous
during
tumor
of
CD44v
epithelia,
have
the first evidence
did
origCD44v
progression
(40,
68).
tumors,
of CD44
provided
a helpful
could
not be fulfilled,
although
to a differing
distinct
primary
to
(18,
In tumors
(35-39).
could
carNon-
dedifferentiation
expression
However,
and
gastric,
cancer
,
renal
in melanoma
nevi,
hoped
metastasis
are one of the more frequent
incidence.
De novo expression
CD44v.
with
appear-
tumor,
cancers,
or
(16).
correlates
universal
lymphoma
formation
epidermis
trans-
system
mammary
the
human
expressed
distinct
v4-v7
in the majority
progression
in melanocytes,
melanomas
exons
and
the
lines
carcinoma,
uteri,
metastasis
isoforms
degree,
7/20/95;
from
rat tumor
on the universal
diagnostic
parameter.
This expectation
because
both nevi as well as melanoma,
Received
may
CD44v
Based
In fact,
be down-regulated
Malignant
that
exons
arose
rat tumors
(colon
lymphoma)
correlate
of
a unique
of leukemia
tumors
the
Hodgkin’s
proven,
of CD44v
was observed
in, e.g.
bladder,
neural,
cervical,
and prostate
types
for some
cinoma
(17).
cancers.
not
growing
variant
as the
variant
isoforms
expression
represent
such
CD44v8-vlO
via the lymphatic
in metastasizing
may
marker
may
which
CD44v
rat,
strictly
of CD44v
that
variant
isoprogression
the
progression
ance
the
of
ap-
exons CD44v3regulated
expres-
but
of locally
to metastasize
in
tissues,
of
(14, 15).
in the variant
splicing
combinations
combinations
exons
combinations
containing
the capacity
10 variant
by alternative
the variant
transfection
by glyco-
exist
multiple
some
it is speculated,
or
functions
interest
eDNA
not
in
CD44vS,
defined
Particular
with
1ev-
lines
of distinct
of exon
in the
vary
which
There
form, in which the variant
3). Regarding
the strictly
exons
the
and skin
metastasized
as malignant
advantage
on specialized
contains
isoforms,
variant
that
29,
benign
be in
in CD44s3
mostly
which
observation
as well
elevated
in melanoma
be inserted
and the keratinocyte
vl0 are inserted
(9-I
ferred
did
can
for
as many lines did not express
variant
exon vlO. Interestingly,
however,
the
accompanied
and
and
nevi,
displayed
of CD44v
melanoma
a linkage
forms
of CD44
primary
would
(1-6).
pre-mRNA
(7, 8). Although
exons
have been documented,
have
metastases,
stained
to
anti-CD44v7-v8,
and
in thick
vS. Expression
few
tested
However,
ex-
metastases
glycoproteins,
structure
which
mela-
v6 and v7 were not detected
on any
Compared
with nevi, expression
20) differed,
inasmuch
isoforms;
in particular,
cytes
of CD44.
up-regulated
Lymph
els of exon
lines
Melanocytes
melanoma
and melanoma
degree
with anti-CD44v5,
anti-CD44v1O.
these
isoform
thick-
and
of
protein
of the
variant
epithelial
vlO by immunohistology
sorting.
The de
increased
of an anchoring
exons,
pear
nevi,
metas-
of varying
were
and skin
cells.
the
tissue.
is a family
sylation
of
metastases,
form
and
cell
variant
node
of
or fluorescence-activated
express
melanomas
15 lymph
and
with
stimulus.
in epidermal
in nevi
melanoma
assumption
compared
a growth
INTRODUCTION
has
of CD44v
in melanocytes,
cutaneous
and lymph
node
65 primary
and
density
exon
epidermal
individual
ness,
metastases
provided
at high
of this
the
node
melanoma,
novo
with
Melanoma’
in lymph
primary
447
Research
S., M. R.,
tases.
exon
and
surrounding
Im
ABSTRACT
In
up-regulated
nevi
line
Progression
R. A.. M. Z.l, Department
of Dermatology.
Rudolf Virchow Hospital,
FU Berlin, Augustenburgerplatz
I. 1000 Berlin 65 [D. 5.1, and
Department
of Applied Genetics,
University
Karlsruhe,
Kaiserstrasse
12, 76128
Karlsruhe
EM. Z.], Germany
variants
was
pression
of Dermatology,
Heidelberg
in Malignant
Cancer
and
patterns
of expres-
metastatic
for differing
malignant
functions
of
isoforms.
The abbreviations
CD44
variant
cence-activated
used are: CD44s.
isoform;
cell
mAb,
sorting;
RT,
standard
form
monoclonal
antibody;
reverse
transcription.
of CD44;
FACS,
CD44v,
fluores-
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer
Research.
448
CD44
Variant
Isoforms
MATERIALS
AND
Antibodies.
antigen,
!gG2a),
of Animal
OJG,
SP4
tihuman
Cell
VFF7
CD44v7,
Cultures,
Porlon
(antihuman
Down,
Salisbury,
91060546;
!gG 1), VFF8
(antihuman
!gGl),
VFF9
(antihuman
7
(an-
used as
VFF16,
and VFF17
were provided
kindly
by Bender
Austria).
They
were derived
from BALB/c
GmbH
(Vienna,
mice immunized
with
The
a
GST-CD44v3-vl0
epitopes
were
lapping
peptides
that
sylation.
The
of CD44
the
specific
A manuscript
in preparation.4
antibodies
variant
in tumor
hidden
these
plot
analysis
not
mAbs
cells.
Melanoma
Lines,
Normal
Human
sues, and Tumor
Typing.
Melanocytes
0.25%
which
trypsin
treatment
medium
and
surgical
and
biopsies.
tissue
1 cm2
43),
(derived
NIS-G
from
(44),
lines
SkMel25
were
growth
from
of
carried
tumors
was
International
used:
Con-
TGIG
Mayer’s
Hialeah,
1 mi
2 mm,
fluent
cultures
were
Before
surface
staining,
2 h at 37#{176}C
in medium
split
after
freshly
containing
trypsinization
trypsinized
antibiotics.
(0. 1%
cells
were
rested
CD44
by the
organs
were
cut into
sections
fluoride,
1 p.g/ml
papain,
1 p.g/ml leupeptin,
and I p.g/ml
were diluted
in L#{228}mmlisample
on a 7.5%
were
polyacrylamide
transferred
386
mti
20%
methanol.
in PBS
containing
I p.g/ml
pepstatin.
Two or
buffer,
boiled
for
gel.
After
elec-
electrophoretically
(300
difluoride
membrane
(DuPont,
activated
in ethanol.
The transfer
glycin,
240
mti
Subsequently,
10%
Tris
the
nonfat
dry
base,
0.006%
membrane
milk
was
powder
and
20. Incubation
with first antibodies
was performed
containing
5% dry milk powder
and I % Tween
20 for 2
h at room
temperature.
at room
Unbound
temperature
the first
optimized
antibodies.
removed
performed
by
protocol
sheets
goat
Dilutions
on standard
PBS
the
After
1%
second
unbound
detection
of the
chemiluminescence
of the manufacturer
were
and
were incubated
antimouse
IgG
of
blots.
washing,
by enhanced
antibodies
in
polyvinylidene
difluoride
radish peroxidase-labeled,
1640
for
between
performed
were
Tween
Con-
FCS.
>25-50%
stained
0-10%
the StatView
computer
of the German
Cancer
survival
Snap-frozen
proteins
blocked
0.5%
trypsin).
-,
was
staining
were
+ +, >75%
(+),
and
x2 test. Correlations
and
endo-
no staining
cells;
cells;
With
to their
blocked,
stained
primary
specificity.
due
heterogenous
score system:
stained
using the
expression
contained
washing
and
>50-75%
resolved
and
Cancer
Research
Center
from xenomelanoma),
FEMX
(48), HS695T
glutamine,
had not been
>10-25%
Blot.
and
at the German
of a malignant
FCS,
of irrelevant
stain-
the
test.
trophoresis,
(established
transplants
10%
controls,
applying
of granulocytes
phenylmethylsulfonyl
in PBS
with
Elec-
and lysed with 150 mi NaCl,
1% Nonidet
P-40,
10 mri TrisHC1 (pH 7.4), 10 mM iodoacetamide,
100 p.M sodium
vanadate,
Cancer
Research
Center
from xenomelanoma),
Mewo
(47),
BS 780
supplemented
according
with an
(Coulter
negative
without
an antibody
which
+,
±,
Western
(46),
Center
(53),
in an intense
faintly
with
counter
For
performed
The results
of the mostly
according
to the following
Research
Center
variant
isoform
SDS,
(50), A375
(51), !F-6 (52),
530Cl
(52). All lines were cultured
in RPMI
streptavidin
were stained
was determined
cells. Statistical
analysis
was done with
program
of the central
computer
program
buffer
MRI-H-22l
were
cell
of the reaction
cells;
cells;
sections
mounted.
cells
Evaluation.
or by using
stained
stained
washing,
was 3-amino-9-ethylcar-
and
S X l0
Fluorescence
was
peroxidase,
at the German
of a malignant
MMLI,
MMLII
(54), and BLM
and
the exception
genous
and
bioti-
FL).
sample
observed.
evaluated
intensively
a second,
After
air dried,
and
washed
with
peroxidase-conjugated
fluorescence-activated
of each
Fixed
temperature
peroxidase
reaction
resulted
sections
were counterstained
hematoxylin,
XL
FACS.
at room
mm
for the enzyme
The
The
EPICS
(established
transplants
(49),
MV3
antibody.
substrate
tronics,
1477
(44),
IgG
mA, 12 h) to a polyvinylidene
Boston,
MA), which had been
(MelJuso;
SkMel28
melanoma),
The
ing
and
for 20 mm
a horseradish
bazole
(Sigma).
red precipitate.
chymostatin,
S pg protein
nitrogen.
sections,
FO-l
(45),
Colo38
Cancer
Research
of a malignant
complex.
log rank
in melanocyte
were obtained
(44),
from
SkMel28;
Ref. 44),
(established
at the German
xenotransplants
by
slides,
and air dried
overnight.
They
10 mm at -20#{176}Cand stored at -20#{176}C
immediately.
melanoma
SkMel5
Tisfrom
tissue
was snap-frozen
in liquid
were cut in 4- to 6-p.m-thick
mounted
on gelatin-coated
were fixed in acetone
for
or immunostained
The following
anti-
Tumor
derived
connective
of the Union
with
antibody
is
First and second
on positive
and
Tissue,
were
from
Typing
to the guidelines
tre Cancer.
Fresh
Sections
of about
Ref.
cleaned
Cells were cultured
(42). Most
tissues
specimens
out according
detail
nM 12-O-tetradecanoylphorbol-l3-acetate
85
overnight.
as described
was
to
constants.5
A biotinylated,
(Sigma
Chemical
Co., St.
negative
foreskin,
lines
the
antimouse
Controls
by glyco-
that
incubated
first antibody.
Slides
were
at room temperature
for 30
incubated
further
in more
revealed
used as the second
antibody.
diluted
in PBS and titrated
newborn
selected
isoforms
Louis, MO) was
antibodies
were
control
for over-
were
describing
similar
binding
antimouse
IgG
were
(55)
were
For FACS
analysis,
to routine
procedures.
recognized
to and competing
epitopes
Scatchard
bodies
exhibited
polyclonal
sheep
protein.
by binding
in ELISA.
for the recognition
guarantee
fusion
identified
nylated
CD44v7-v8,
(antihuman
CD44v1O,
!gGl)
were
mAbs SFF2, VFF7,
VFF8,
VFF9,
sections
with the
incubated
; European
number
CD44v6,
VFF1
dried
melanosomal
IgGl
catalogue
CD44s,
IgG 1 ),
IgG2b),
and VFF16
first antibodies.
The
(antihuman
CD44s,
Kingdom;
(antihuman
IgGl),
Ta99
(antihuman
United
41 ), SFF2
CD44vS,
mAbs
25-32
Wilts
Immunohistochemistry
METHODS
The
Collection
Ref.
in Melanoma
removed
Tween
by
20.
The
with the horseas described
for
antibody
had
been
antibody
had
been
specific
proteins
was
according
to the
(Amersham,
Braunschweig,
Ger-
many).
mRNA
cation,
E. Patzelt and G. Adolf, Human CD44 variant isoform-specific
clonal antibodies:
preparation
and characterization,
manuscript
4
M. ZOller,
Southern
frozen-tissues
acid
phenol
method
unpublished
finding.
total
RNA
cDNA
Blot.
or cultured
was synthesized
aration.
‘
monoin prep-
Preparation,
and
of
cells
primer
was
PCR
extracted
by the guanidine
Chomczynski
and subjected
and oligo(dT)
Preparation,
mRNA
and
to PCR
snap
isothiocyanate/
Sacchi
amplification
for synthesis
Amplififrom
(56).
using
of the first
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer
Research.
eDNA
2 p.g
strand.
Clinical
Table I
Expression
of CD44
variant
malignant
exons
melanoma
(n =
Nevi
13)
of CD44
(Fig.
1). Furthermore,
serial
sections
with
(% of tumors)
s
vS
v6
v7
v7-v8
vl0
100
100
100
77
78
0
0
0
0
92
86
100
0
0
100
85
68
73
100
85
0
0
47
90
tissue
Primary tumor (n = 65)
Lymph node metastases
(n
15)
Skin metastases
(n = 39)
and metastatic
tissue
Expression
Melanoma
in primary
with
any
nevi
exon-specific
amplification.
Amplification
3’ oligos
for
variant-specific
29
expressed
were
labeled
EDTA,
was
were
performed
to CD44s-
by the S ‘ end-labeling
7% SDS, and 0.5
(inverse),
v2
AlGA;
GAC
CAC;
GCA
and
AGC
ATG
v4, ATG
v5, ATG
CCA
CD44v-specific
AAT
in nevi
GAA
TGGG;
CAG
CTC
ATA
CCA
GCC;
melanoma
evaluated
tumors
this
cells
twice
>25-50%,
the variant
and CD44vlO
with
1 spindle
and
metastases,
varying
cell),
nevi, primary
tumors,
the tissue specimens
Expression
of the
90% of nevi
CD44v7-v8,
seen
node
less
frequently
metastases;
anti-CD44v7-v8.
<50%
However,
anti-CD44v5.
anoma,
It should
be mentioned
and metastases
with
antibodies
instances.
did not reveal
These findings
was
expressthat
down-regulated
but up-regulated
In comparison
CD44v I 0 was up-regulated
expression
of CD44v5
was
cells
2) revealed
in
in thick (>0.7
with
primary
in
thin,
mm), primelanoma
further
in skin metastases,
whereas
up-regulated
in lymph
node metas-
tases.
To obtain
of expression
further
of
evidence
distinct
for potential
CD44
clinical
isoforms,
relevance
patients
were
also
GCT
GTC
tumors
and
the
CAG
CCI
tumors
and
detection
AG AAT
TCG
Expression
of none of the CD44v
exons
correlated
with the
grading
of the examined
tumors.
The statistical
analysis
of a
vlO
(inverse),
GTf
mice
pads.
were
GGAA.
received
S X 106
Tumor
growth
sacrificed
when
the longest
was
the
duration
for
according
possible
and Variant
Melanoma.
Isoforms
in
Expression
CD44v6,
in 13 nevi,
(7 intradermal,
tumors
variant
5 epidermal,
(38
correlation
observation
node
1). Although
period
80-
melanoma
metastases
node
and
stained
metastases
Lines.
CD44
lymph
with
stained
that staining
of nevi, primary
melanti-CD44
variant
isoform-specific
a uniform
pattern
of staining
in most
are illustrated
for a primary
melanoma
sion
estimate
the appear-
specimens
expressed
of CD44v
high
CD44
some
by sugar
chains
due to glycosylation.
during
A further
themselves
To prove
melanoma
interesting
ob-
expression
of metastasis.
Isoforms
as variant
epitopes
our
by up-
of CD44v
localization
Variant
supported
is accompanied
the pattern
as well
expression,
parameters,
isoforms.
that
on the organ
change
of the
deaths.
tissue
melanoma
progression
Therefore,
patterns
and
However,
clinical
CD44
standard
appearances
the patients’
expression
of their
with
the finding
of
primary
not shown).
melanoma
depending
Expression
hidden
changes
antiwas
malignant
was
first
and/or
months.
died
(data
of distinct
servation
of
variable:
of skin
was
that
regulation
glycosylated.
extent
with anti-CD44v5,
staining
with CD44vlO
had
of their
was hampered
by the low numbers
metastases
or had died during our
our semiquantitative
assumption
the
CD44v
of 6-24
who
as the correlation
all
CD44s,
none
or anti-CD44v7.
between
of CD44v5
as well
13
15 lymph
between
grading
of metastases
ance of a metastasis
or exitus
of patients
who had developed
varied
superficial,
(Table
exons
CD44v7,
7 of them
to the histological
intervals
of all patients
all lymph
with
of tumor
(Table
CCG
metastases
in primary
the tissue specimens
were
percentage
(>10-25%,
TCA
and metastases
expressed
stained
with anti-CD44v6
and
CD44v1O
a
(S. S. and
GAT
histopathology),
remaining
nevi,
observers
ason
AAT
primary
stained
to some
and anti-CD44v1O;
analysis
this
ATG
histology
and 37 cutaneous
>75-100%)
primary
melanoma
mary
melanoma.
iT!’
months.
14 of less frequent
and
check
evaluated
v7, ATG
exons
CD44v5,
was evaluated
65
independent
2). Statistical
to
Thus,
congenital
nodular,
(Fig.
To
was
was
isoforms
grouped
of CD44 Standard
Nevi, and Malignant
and
and
>50-75%,
ing CD44
RESULTS
of CD44s
CD44v7-v8,
CD44
D. S.). Using a microscopic
estimate,
grouped
according
to the approximate
levels
Expression
Melanocytes,
of distinct
ATG
and
>3
melanoma
isoforms
by two
expres-
metastatic
GAA
of 0.5 cm;
was
basis
a
ACA
GAC AT!’ AGA
Assay.
nu/nu
a diameter
to occur
1TG
GCT
semiquantitative
the heterogeneous
progression.
of CD44
isoforms,
TAG
injected
into foot
per week.
Animals
reached
growth
GGA
GIG
CGG
variant
but not
not shown).
ATG
v6 (inverse),
GTC
GAG
liT
CCA
CD44
and
in melanoma
expression
of anti-CD44
and malignant
transformavariant
isoforms
were not
that expression
be involved
sumption,
stained
anti-CD44s
(data
However,
and primary
indicating
panel
with
express
in melanocytes.
patterns
comparison
CAA
CCA
ACA
CCG
G1T
CGA 1TG AT!’
In Vivo Metastasis
probes
were used (7):
ATG GACC;
TFG
GGC
TCG
ICC
GAT
CTGC;
CAA
AAT
CCT
GAA
5’ and
procedure
in church
buffer (1 mi
sodium
phosphate
buffer,
pH 7.2;
M
CAA
(inverse),
AAG
the
were transferred
to
Hilden,
Germany)
Ref. 57). The following
CD44 oligonucleotides
5’-oligo,
CAG ACC TGC CCA ATG CCT
TGCC;
for
at 66#{176}C
with
indicated,
PCR products
Plus membrane
(Quiagen,
hybridized
3’-oligo
used
cycles
and at 59-67#{176}C for 35 cycles
with
oligos.
PCR products
were analyzed
on a 1 %
agarose
gel. Where
a Quiabrane
Nylon
and
primers
also
the
449
When
double
antibodies
linkage
between
CD44v
expression
tion was excluded,
although
CD44
may
variant
anti-CD44v
did
were
stained
Research
differently.
skin
and
double
of the other
Because
behaved
human
antibody
melanocytes
suspicious,
or
black
an antimelanocyte
antibodies,
sion
Standard
melanocytes
of normal
Cancer
in
isoforms
of the
Melanoma
are heavily
molecule
can
be
or by conformational
that the CD44
expresprogression,
it was
nec-
essary to compare
surface
staining
with the presence
of CD44v
proteins
by Western
blotting.
In the first instance,
the comparative analysis
mating
signals
staining
was done with melanoma
lines to avoid
of the surrounding
skin. Surprisingly,
of melanoma
cell
lines
differed
considerably
contamsurface
from
of tumor tissue (Fig. 3). Except
for MMLI!,
all melanoma
as well as melanocytes
stained
brightly
with anti-CD44s.
only one cell line (BLM)
stained
with anti-CD44v7-v8
anti-CD44v1O,
and CD44v6.
and only a few lines stained
with
Different
from the in situ staining,
that
lines
But,
and
anti-CD44v5
a faint reac-
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer
Research.
450
CD44
Variant
Isoforms
in Melanoma
v----
‘
.
-
-#{149}4_’_
-
‘f;’
:
.
._i,
4‘
-
:,
-
Expression
Fig.
I
with
hematoxylin
and
anti-CD44v7-v8
stained
of CD44s
(G).
with
the
of
CD44vlO
(A),
eosin
and
cultured
melanocytes
was
noted.
The Western
blot
3), which implies
(Table
epitopes
antibody,
recognized
X
extracts
cells
had
been
in a
with
control
anti-CD44s,
anti-CD44v6
and
anti-
analysis
revealed
that in melanoma
comparable
results
cell lines, none of the
of mAbs
was hidden.
node
replaced
by the tumor
CD44v6
metastases,
protein
in which
masses,
was
Expres-
all lymphoid
were
blotting,
shown).
antibody
Hence,
the epitope
recognized
by our
was accessible
in vitro but was hidden
clearly
screened
present
by
(data
not
anti-CD44v6
in melanoma
tissue.
and
second
melanoma
exon
skin
feature
cell
at variance
lines
was
was expressed
strongly
metastases,
but hardly
except
MMLII
and
4).
Two
Mewo,
between
melanoma
the expression
in metastatic
in melanoma
were
derived
tissue
of CD44vlO.
tissue,
cell
from
This
in particular,
lines,
which,
metastatic
v6-specific
tissue.
probes;
blotting
These
findings
imply
in vitro
culture
The
the
sion
mRNA
node
amplified
by RT-PCR
with
CD44s-
and CD44v-specific
was
primers
nude
lines
mouse,
exon
level
uncovered
may
exon
v7-
by exon
by
Southern
v6-,
and,
was
particlines.
not translated
two
v6);
lines
phenomena:
may
be
at
and
(a) expres-
by surface
staining
and (b) more
variants
exons
melanoma
due
importantly,
regulated
at the
vl0).
Melanoma
from
of CD44v
in
be ignored
(exon
splice
(exon
metastatic
BLM,
whereas
exon
amplification
vS-,
mRNA
vS-
by
of the melanoma
of expression
mRNA
As mentioned
(Mewo,
by
RT-PCR
in most
analysis
the
CD44v5-positive
the
which
exon
recognized
confirmed
after
CD44v1O
changes
level
rarely
was
were
Again,
by
recognized
conditions.
of CD44
translational
mRNA
eDNA,
results
that
epitopes
to conformational
established
into
signal)
present
metastases
of distinct
were
transcribed
was
versus
expression
recognized
probes
comparative
protein
lymph
was
probe.
vl0
was
(strong
The
a CD44s-specific
Mouse.
and
-
recognized
signal)
exon-specific
Nude
extracted
one
and one
ularly,
under
were
(weak
probes.
with
To answer
the question
of whether
exon vlO was spliced
out at
the pre-mRNA
level under
the in vitro culture
conditions,
or
whether
the spliced
mRNA
was not translated
into protein,
was
fragments
one
probes;
specific
with
PCR
probes;
vlO-specific
lines,
however,
was
in melanoma
tissue.
Western
The
(Fig.
specific
of lymph
-
200.
with
by our panel
-
malignant
melanoma.
Frozen sections of a primary
melanoma
were stained
(TA99;
B), anti-CD44s
(C), anti-CD44v5
(D), anti-CD44v6
(E), anti-CD44v7
(F),
(second antibody only). Tumor areas are indicated by arrowheads
(in G). The tumor
and partially with anti-CD44v5,
anti-CD44v7-v8,
and anti-CD44vlO.
It did not stain
primary
antibody
sion of CD44v6
in some melanoma
cell
contrary
to the apparent
absence
of CD44v6
When
-
isoforms
1, negative
(H).
and anti-CD44v7.
also
variant
a melanocyte-specific
anti-CD44vlO
melanocyte-specific
with anti-CD44v6
tivity
and CD44
MV3,
others
Lines
already,
tissue.
and
(IF-6,
Metastasize
most
However,
A375)
S3OCl,
in the
of melanoma
only
metastasize
SkMel5,
lines
some
of
in the
SkMel25,
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer
Research.
Clinical
CD44v5
CD44v7-v8
Cancer
Research
451
CD44vIO
100
90
80
70
C
0
E
U
a
60
Il)
4,
In
In
50
4,
40
30
20
10
0
.;
4,
z4,
Fig. 2
Quantitation
(n = 15) and thick
anti-CD44v7-v8,
z
.E
#{149}
.C
.
:
.C
.iIn
(n
and
of cells were stained,
;
C
.X
-
The
percentages
of tissue
2
Changes
in th e expression
Thin
with
Nevi
P values
samples,
profile
of CD44
variant
isoforms
variant
during
isoform
melanoma
vS
v7-v8
ns
vlO
0.97
0.0004
vS
v7-v8
ns
ns
I
0.0067
node metastasis
vS
v7-v8
vl0
vS
ns
0.99
ns
0.038
I
I
0.016
0.007
0.01 1
v7-v8
vlO
ns
0.046
significant.
in lymph
node
of whether
metastases.
surface
These
expression
melanomas
metastasize
personal
communication
in general
in the nude
The
hypothesis
observations
raised
cell
lines
into
of CD44v5
in ma-
was
known
astatic
capacity.
which
differed
tion
pads.
of the tumor
two
The
controlled
We
to metastasize;
and
correlates
the
one
latter
in expression
by injection
choose
(SKMe128
cells,
with
capacity
to
mouse.
was
foot
metastasize;
5 Matzku,
vlO
0.053
Lymph
of CD44v5
6
melanoma
0.18
lignant
question
_
progression
Thick
and SkMel28)
do not (52, 54)6
To our surprise,
all cell lines that
are known
to metastasize
expressed
high levels
of CD44v5.
Furthermore,
immunohistochemistry
had revealed
up-regulation
the
-
In
expression
melanoma
ns”
0.99
Primary melanoma
P values
not
.C
and metastatic
melanoma
tissue. Frozen sections of nevi (n = 1 3), thin
15), and skin metastases
(n = 39) were stained with anti-CD44vS,
where
>10-25%
(Li ), >25-50%
(11 ), >50-75%
(U ), and >75-100%
( U)
Skin metastasis
ns,
.
(n
CD44
a
.
4,
In
Z
C
.E
.
are shown.
Table
Compared
.;
.-
z
of expression
of CD44 variant isoforms in primary
50) primary
melanoma,
lymph node metastases
anti-CD44vlO.
Z
C
.
two
four
of melanoma
lines:
one
(SkMel25)
was
known
and
had
unknown
were
1477)
sublines
of CD44vS
all animals
cell
were
of one
(Table
surveyed
(BLM)
not
to
met-
melanoma,
4). After
injec-
for local
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer
Research.
and
452
CD44
Variant
Isoforms
in Melanoma
100
1
.
.
.
90
.
.
.
.
.
.
.
.
.
.
a
a
S
.
.
80
...
70
0
a
.
.
a,
Os
60
U
V
50-
.E
#{149}v5
.
4,
‘4
a
ov6
.0.
#{149}v7
0
a
.
ov7-v8
AVIO
I.-
;
40
A
30
U
0
a
0
20
A.
0
0
0
10
0
U
A
U
#{149}
A
U
6
$
U
A
p
#{149}
S
0
#{149}ai
#{176}
CO
(
I-#{149}
U
0
Fig.
of
3
Staining
anti-CD44
of melanoma
antibodies
lines
with
described
Table 3
LL
Cl)
I
.
C,)
MMLII
CD44s
FACS
CD44v5
FACS
Western
Southern
CD44v6
FACS
Western
Southern
CD44v1O
FACS
Western
Southern
a
See
‘
CD44-specific
antibodies.
Cultured
of stained
pecific
betw een CD44v-s
surfac e
staining,
of protein,
expression
with the panel
and mRNA
in melanoma
lines
lines
SkMel28
Co1o38
530C1
Mewo
1477
BLM
++
++
++
++
++
++
++
++
++
±
++
++
nt
++
++
++
++
++
++
nt
±
++
++
++
nt
++
++
++
++
++
++
-
-
-
(+)
±
±
±
(+)
(+)
(+)
++
±
(+)
(+)
+
±
nt
±
-
+
nt
-
±
±
+
nt
±
+
+
+ +
+
+ +
-
-
-
±
±
±
±
(+)
(+)
++
++
-
-
±
nt
±
±
(+)
+
+
+
nt
-
-
+
+ +
nt
±
+ +
+
+
±
+
-
-
-
-
-
±
-
-
-
-
-
±
nt
-
-
-
±
-
±
nt
±
+
nt
+
+ +
+ +
+
+
-
‘Materials
and Methods’
‘
of about
months.
0.5 cm after
2-3
development
and
lines (n = 20) were stained
FEMX
All
slow,
roscopically,
and melanoma
melanocytes
cells is shown.
HS695T
-
The
“
Cl)
C.)
Mel Juso
metastatic
tumor
growth.
mors. Tumor
growth
was
3 months.
.
SkMel25
+“
Western
Southern
0
0
!.
Melanoma
Exon
.2
I
C/)
in Fig. 1. The percentage
Corn parison
?z
.
i)
by
seeding
for explanation
of symbols.
All mice
dispersed
were
was
lung
sacrificed
evaluated
and
lymph
(+)
nt, not tested.
animals
developed
primary
tubut tumors
did reach a diameter
of metastases
+ +
++
lines
lung
lacking
expression
metastases.
After
after
CD44v5,
mac-
most
instances,
the lymph
node
metastases
and
immigration
of tumor
cells
was
node
tissues
in culture
flasks
under conditions
allowing
for the survival of tumor cells. None of the mice that had received
the cell
some,
of exon vS developed
transplantation
of cell
although
not all, mice
lymph
node or
lines expressing
developed
verified
metastases.
were
only
In
rather
small,
by
in vitro
culture
of dispersed
lymph
nodes.
In 8 of 1 1 mice, melanoma
cells grew in the tissue culture
explant
of the draining
lymph
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer
Research.
Clinical
-
Cancer
Research
453
-
.
-,-
r_
c:
.
U-
c
GAPDH
Fig.
Expression
4
variant
isoform
of
mRNA
CD44
on
mela-
noma
lines.
The presence
of
CD44 variant isoform mRNA in
melanoma
lines was tested at the
eDNA level after RT of mRNA
and amplification
of eDNA by
RT-PCR.
eDNA was amplified
with CD44s-,
CD44vS-, CD44v6-,
CD44v7-,
and
CD44v1O-specific probes.
Left to right, MMLII
(no surface
staining),
Colo38,
HS695T,
Meliuso,
Mewo,
SkMel25,
SkMel28,
1477,
:
.
within
scopically.
The
anoma
lung,
that
capacity
indicated
progression.
v6
Exon
v7
Exon
vlO
_.
-
In 6 animals,
where
3-10
melanoma
cells
were
detected
nodules
had
even
DISCUSSION
macroAfter
finding
metastasizing
melanomas
1-2 weeks.
in the
Exon
530C1.
-
settled
v5
-.
BLM, size standard, and negative
control.
The glyceraldehyde-3phosphate
dehydrogenase
control
of the RT-PCR
is included.
For
RT-PCR conditions
and primers,
see ‘ ‘Materials
and Methods.”
node
Exon
expression
of melanoma
that
CD44v5
of CD44vS
cell lines
is possibly
correlated
with
as well
as primary
relevant
the
in mel-
variant
rat
tumor
a striking
isoforms,
model
correlation
particularly
(16),
many
between
exon
studies
expression
v6, had been
have
explore
in which
human
tumor
systems
may exist and could be used for diagnosis
been
of CD44
discovered
in a
performed
a similar
or therapy
correlation
(reviewed
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer
Research.
to
454
CD44
Variant
Table
Isoforms
4
in Melanoma
Metastatic
growth
of
melanoma
in nu/nu
lines
apparently
particular
The
mice
Metastases
Melanoma
(5 X 10”)
line
CD44v5
SkMel25
Local
Lymph
tumor
node
-
SkMe128
(+ )
Lung
(no.
of
5/5
5/5
0/5
0/5
0/5
0/5
noma
nodules)
1477
+
6/6
4/6
3/6
+ +
5/5
4/5
3/5 (3-10)
17, 58, and
melanoma.
increasing
rapidly.
59).
The
We were
incidence
However,
tumor
ing features.
ticular:
(a)
revealed
tumors;
primary
melanomas
up-regulation
of CD44
Finally,
we
findings
would
using
vS
to
at least
However,
CD44v5,
and
and
ofCD44
skin
in lymph
most nevi,
CD44v7-v8,
node
briefly
versus
in vivo,
malignant
exclude
transformation
a linkage
in
(c)
the
metastasis.
on
Western
CD44
variant
Although
nevi
as well
variant
isoforms,
antibodies.
Even
but
expression
expression
cell lines.
patterns
were
There are two
size
that
the
observation
both
glycosylated
may
as malignant
neither
more
melanomas.
expression
they
and
did
tumor
heavily
The
a mechanistic
(3-5),
and
first
may
chain
the melanoma
metastases,
directly
or ligand
The
pro-
could be detected
by hidden
epitopes.
even
mAB-recognizing,
epitopes
can reveal
rather
neighboring
differing
was
tastases
could
7
S.
manuscript
by Western
We would
that
either
due
some
mela-
contact
,
to
of
melanomas
and
mAB,
although
blotting,
could
like to add that
or overlapping
patterns.7
Thereof CD44v
whenever
with
whereas
contact
form
may
cell
studies
In view
facilitate
carcinomas
lose
acts
likely
and
that
tumor
homing
cell
cell
expression
It is unlikely
that
to the insensitivity
nohistology
and
of
via homotypic
CD44
cells
penetration
carnode
variant
transfected
isowith
binding
and
by the chon-
of the
All these
on cells
that extight
june-
cells.
Considering
the loss of CD44v1O
node metastases
and melanoma
cell lines,
was detected
unexpectedly
in many
mela-
lines.
due
facilitates
into
lymph
CD44vlO-cDNA
grow in aggregates.’#{176} Homotypic
aggregate
formation
are supposed
to be promoted
of CD44vlO
and
of further
(c) squamous
most
me-
brightly
with
as lymphocytes
particularly,
revealed
homodimers,9
skin
metastases
stains
as well
and,
CD44v1O
(d)
lines.
We
of nevi
and
node
CD44vlO;8
the subcutis
cross-linking
only
in the majority
epithelium
lymphomas
express
and
noma
changes
e.g.
melanoma
in lymph
CD44v1O
squamous
is
with
M. R#{246}sel,
W. Tilgen, C. Claas, and M. Z#{246}ller,
Distinct
profiles
of CD44
variant exon 10 are tissue related,
in preparation.
of
(69);
pression
At least some of the discrepant
findings
about CD44
isoform
expression,
e.g., in colorectal
cancer
(29, 61),
Seiter,
skin
of
observation
that
infiltrating
noted
in melanoma
the
epidermal
(b) cutaneous
binding
easily
of distinct
or malignant
cells,
in thick
partially
we assume
metastases
forms
lost
be detected
the
was
further
was
hardly
cinomas
Western
PCR amplification,
a relatively
low
we
missed
of the detection
blot)
compared
because
detection
blotting
and immunohistology.
ulate CD44vlO
expression
the
CD44vlO
methods
with
(immu-
Southern
blots
we had chosen
a PCR protocol
level, quite similar
to Western
The mechanism
at the mRNA
level,
that could reghowever,
is still
unknown.
The
anoma
analysis
revealed
melanoma
cell
of lymph
node
a significant
lines
metastases
up-regulation
expressing
exon
vS
of malignant
mel-
of CD44vS.
Also,
metastasized
nude mouse
via the lymphatic
system.
Interestingly,
isolated
from the draining
lymph
nodes
of nude
N. Wagner,
S
and
M. Goos,
exhibit
glycosylation
but
e.g.,
fore, we suggest
that immunohistological
definitions
expression
should
be verified
by Western
blotting
possible.
variant
as
melanoma
expression
by CD44v1O,
of CD44v1O
that,
inaccessible
of a nevi
be facilitated
up-regulated
CD44
epitopes
staining
as well
and
droitin-4-sulfate
side chains carried
by CD44v 10 (64).
features,
including
the loss of CD44v1O
expression
leaving
the epidermal
context,
support
the assumption
melanoma
we emphadeals
Thus,
cells
on the neighbouring
may
view.
cell lines, but none of the primary
stained
with
the anti-CD44v6
CD44v6
protein
well be explained
cells
of
or by conformational
binding.
and
embed-
tissue
Nevi
activity.
on individual
Expression
and
after
with
glycosylation
63).
de-
progression,
facilitates
in foreign
62,
are
were
with Langerhans
cells may require
expression
of CD44v5.
are trying to verify the validity
of this hypothesis
currently.
backbone
by a sugar
in tumor
cells
(16,
tumors
melanomas
isoforms
of CD44v
tumor
nutrients
depend
epithelial
protein
can become
involved
Most
and
variant
expression
of proliferative
exons
noma
CD44 to hyaluronic
acid varies with the activation
status of the
cell (60). Thus, any epitope
for a mAb raised against
the protein
of the molecule
of
stage
binding:
not
expressed
explanation
is known
that
tions
of epidermal
protein
in lymph
CD44vlO
mRNA
point
it
supply
CD44vlO
uniformly
with antithe same
unstable
noted with in vitro-cultured
possible
explanations,
and
be relevant.
from
melanoma
stained
surprising,
of metastasizing
their
infiltrating
gression.
CD44
CD44v
ding
skin:
(a)
CD44vlO;
irrespective
of their histology,
and CD44vlO.
The same ac-
CD44v
CD44
as being
is evidence
evidence,
disparate
blotting.
did not express
in melanoma,
between
vl0
and
counted
for primary
and metastatic
malignant
These
findings
excluded
a linkage
between
CD44v
and
me-
interest-
exon
metastasis;
comment
immunohistology
Melanocytes,
isoforms.
expressed
exon
like
early
should
be discussed
in parCD44v
expression
pattern
of
and
tissue.
nevi
CD44v
peculiar
(b) the up-regulation
originally
there
melanoma.
whereas
early
an
melanomas
some
in foreign
topographic
and
Thus,
in malignant
thereof
Three
observations
the heterogenous
individual
thick,
expression
malignant
of mela-
the
ma-
diagnosis
of metastatic
spread
could
facilitate
the
of curative
therapy
greatly.
Our analysis
of CD44
derived
and
of origin,
on
Furthermore,
reliable
likeliness
isoform
tissue
in their
staining
relies
to a
arising
in foreign
tissue may well require
specialized
molecules,
such as CD44v,
for embedding
and the supply
of nutrients
at an
at an early
tastases
of development.
of nevi
arise
diverse
and
epitopes
fre-
melanomas
immunogenic.
hypothetical
of some
is diagnosed
lignant
are
is still
peculiarities
scribed
quently
variant
stage
(1-8)
especially
interested
in
of this tumor
has been
the
cells
embedded
BLM
in Refs.
malignant
are due to the inaccessibility
anti-CD44v
mAB.
second
explanation
for the
a unique
H. P. Bertsch,
Malignant
pattern
and
of
C. Wagner,
reactive
CD44
G. Nussbaum,
cutaneous
splice
variants,
lymphoid
M. Z#{246}ller,
cell
submitted
cation.
9
tO
in the
tumor cells
mice stained
Sleeman, personal communication.
M. Ztiller, unpublished
finding.
J.
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer
Research.
infiltrates
for publi-
Clinical
brightly
with
anoma
anti-CD44v5,
cell
lines
whereas
did not gain
only
CD44v5
vivo
passage
(data
not
these
findings
could
be a growth-promoting
in lymphatic
tissue.
and CD44v6
bind
cytokines
expansion
of
(65,
in addition,
growth
fection
66),
of CD44v-negative
Although
nevi
variant
CD44
CD44v1O
and
pointed
CD44vS
toward
skin
CD44v1O,
the
working
hypothesis
propriate
recruitment
and
and
data
also
that
tumor
currently
lines
lymph
with
strong
of physiological
for
CD44v5.
CD44v
ex-
the physioof CD44v5
support
relies
programs
of
for
on the
our
map-
mAbs,
Cancer
and
Research
L.
Center)
who kindly
Edler
provided
(Department
of
for the statistical
the anti-CD44s
Biostatistics,
and
German
analysis.
mun.,
13.
14.
L. J., Nakache, M., and Butcher, E. C. Monoclonal
antibodies
lymphocyte
homing receptors define a novel class of adhesion
on diverse cell types. J. Cell Biol., 109: 927-937,
1989.
3. Lokeshwar,
V. B., and Bourguignon,
L. Y. W. Post-translational
protein modification
and expression
of ankyrin-binding
site(s) in GP85
(Pgp-1/CD44)
and its biosynthetic
precursors
during T-lymphoma
membrane biosynthesis.
J. Biol. Chem., 266: 17983-17989,
1991.
4. Camp R. L., Kraus, 1. A., and Pure, E. Variations
in the cytoskeletal
interaction
and posttranslational
modification
of the CD44 homing
receptor in macrophages.
J. Cell Biol., 115: 1283-1292,
1991.
5. Omary,
M. B., Trowbridge,
I. S., Letarte, M., Kagnoff, M. F., and
C. M. Structural
heterogeneity
of human Pgp-l and its relationwith
p85.
Immunogenetics,
27: 460-464,
1988.
Isacke,
6. Dougherty,
G. J., Lansdorp,
P. M., Cooper, D. L., and Humphries,
R. K. Molecular cloning of CD44R1 and CD44R2,
two novel isoforms
of the human CD44 lymphocyte
“homing”
receptor expressed by hemopoietic cells. J. Exp. Med.,
174: 1-5, 1991.
0.
R.,
Bell,
M.
V.,
Jackson,
D.
0.,
Cornelis,
F. B.,
Gerth,
U., and Bell, J. I, Genomic
structure of DNA encoding the lymphocyte
homing receptor CD44 reveals at least 12 alternatively
spliced exons.
Proc. Natl. Acad. Sci. USA, 89: 12160-12164,
1992.
8. Tolg, C., Hofmann,
M., Herrlich, P., and Ponta, H. Splicing choice
from ten variant exons establishes
CD44 variability.
Nucleic Acids Res.,
21: 1225-1229,
1993.
9. Brown, T. A., Bouchard,
W. G. Human keratinocytes
as
a heparan-sulfate
exons.
intrinsic
J. Cell Biol.,
113:
T., St. John, 1., Wayner, E., and Carter,
express a new CD44 core protein (CD44E)
membrane
207-221,
proteoglycan
with
additional
1991.
15.
J.
and
Butcher,
E.
C.
Identification
Liao,
(Cold
H.
X.,
(CD44):
Spring
and
multiple
L.
184:
that
and non-
transmembrane
forms.
Can-
1991.
of isoforms
Immunol.,
The
multiple
347-350,
a multitude
Microbiol.
mRNA
1990.
K.
functions,
3:
Harbor),
CD44:
Patton,
of
in lymphoid
with
47-63,
diverse
functions.
1993.
16. Gtinthert,
U., Hofmann,
M., Rudy, W., Reber, S., Zdller,
M.,
Haussmannm
I., Matzku, S., Wenzel, A., Ponta, H., and Herrlich, P. A
new variant of glycoprotein
CD44 confers metastatic
potential
to rat
carcinoma
cells.
Cell,
65: 13-24,
1991.
17.
ZOller,
M.,
114-122,
and
Kaufmann,
M.
CD44
and
metastasis.
17:
Onkologie,
1994.
aggressive
non-Hodgkin’s
rat metastasis-associated
1993.
lymphomas
variant
G. R., van den Berg,
human lymphocytes
express
of CD44.
a homologue
J. Exp. Med.,
of the
897-904,
177:
19. Kuppner, M. C., Van Meir, E., Gauthier, T., Hamou, M. F., and De
Tribolet,
N. Differential
expression
of the CD44 molecule
in human
tumours.
are
A.
C.,
expressed
Cancer
21.
Int.
in
Van
Meir,
human
Res., 53:
E.
brain
G.
1992.
N., Jaufeerally,
R., Hofmann,
Metastasis-related
variant
G.
metastases
5345-5349,
but
not
in
M.,
CD44
glioblastomas.
1993.
J., Akinwunmi,
Differential
binding
port, 4: 259-262,
J., Ognjenovic,
of anti-CD44
N.,
on human
and
gliomas
Rogers,
in vitro.
J. P.
Neurore-
1993.
Cannistra,
S. A., Kansas,
and Ouensmeier,
C. Binding
sothelium
in vitro is partly
G. S., Niloff, J., DeFranzo,
B., Kim, Y.,
of ovarian cancer cells to peritoneal
me-
22.
3830-3838,
572-577,
M. F., de Iribolet,
and
Pilkington,
50:
J. Cancer,
Li, H., Hamou,
mediated
by
CD44H.
Cancer
53:
Res.,
1993.
Heider, K. H., Hofmann,
M., Horst, E., van den Berg, F., Ponta, H.,
P., and Pals, S. I. A human homologue
of the rat metastasisassociated
variant
of CD44
is expressed
in colorectal
carcinomas
and
adenomatous
polyps. J. Cell Biol., 120: 227-233,
1993.
23.
Herrlich,
24. Kim, J. C., Ishii, S., Ford, R., Thomas,
J. M. Epitopes
for homotypic
binding
antigen
also
Proc.
25.
participate
Am. Assoc.
Jothy,
S.,
of
carcinoma.
Proc.
26.
Tanabe,
in
Cancer
McClure,
expression
K.
K.,
725-726,
1993.
Ellis,
to
L.
M.,
in colon
and
GUnthert,
Saya,
carcinomas
variants
proteins.
H.
U.
in
34: 553.
Res.,
and
matrix
1993.
splice
Cancer
G., Jr., and Jessup,
carcinoembryonic
extracellular
L.,
RNA
Assoc.
P., Steele,
of human
197,
LeDuy,
messenger
Am.
molecule
adhesion
Res., 34:
D.,
CD44
adhesion
Selective
human
colon
1993.
Expression
of
and metastases.
CD44RI
Lancet,
341:
Mayer, B., Jauch, K. W., Gtinthert,
U., Figdor, C. G., Schildberg,
F. W., Funke, I., and Johnson, J. P. De novo expression
of CD44 and
survival
in gastric
cancer.
Lancet,
342: 1019-1022,
1993.
27.
Heider,
K. H., D#{228}mmrich, J., Skroch-Angel,
P., Mtiller-Hermelink,
H. K., Vollmers,
H. P., Herrlich, P., and Ponta, H. Different expression
of CD44 splice variants
in intestinal
and diffuse type human gastric carcinomas and normal gastric mucosa. Cancer Res., 53: 4197-4203,
1993.
28.
of CD44
10:
progression.
1991.
F.,
G#{252}nthert, U.
10. Stamenkovic,
L., Aruffo, A., Amiot, M., and Seed, B. The hematopoietic
and epithelial
forms of CD44 are distinct polypeptides
with
different
adhesion
potentials
for hyaluronate-bearing
cells. EMBO J.,
343-348,
keratinocytes.
1992.
1992.
A.,
receptor
Curr. lop.
Diserens,
7. Screaton,
B.
Cells
1. Jalkanen,
ship
L.
Haynes,
20.
to human
molecules
on
proteoglycan
encodes an alternative
form of H-CAM (CD44)
lymphoid tissues. Immunogenetics,
32: 389-397,
brain
2. Picker,
L. M. Identification
surface
374-380,
182: 569-578,
Goldstein,
REFERENCES
S., Bargatze,
R. F., de los Toyos, J., and Butcher, E. C. A
lymphoid
cell surface protein involved in endothelial
cell recognition
and lymphocyte
homing
in man. Eur. J. Immunol.,
16: 1195-1202,
1986.
455
R. H., and Milstone,
a cell
99:
Dermatol.,
Research
12. Cooper,
D. L., Dougherty,
G., Ham, H. J., Jackson,
S., Baptist,
E. W., Byers, J., Datta, A., Phillips, 0., and Isola, N. R. The complex
CD44 transcriptional
unit:
alternative
splicing
of three
internal
exons
generates
the epithelial
form of CD44. Biochem.
Biophys.
Res. Corn-
and
We thank Drs. E. Patzelt,
of
18. Koopman,
G., Heider, K. H., Horst, E., Adolf,
F., Ponta, H., Herrlich, P., and Pals, S. I. Activated
(67).
ACKNOWLEDGMENTS
anti-CD44v
J. G., Bretton,
characterization
Invest.
cer
metastases
two
1 1. Haggerty,
and
hyaluronate
patterns
account
progression
in
expressed
functions
provide
cells
by trans-
node
into
supposed
the
well
of CD44v5
of these
Taking
pattern
tumor
expression
importance
them
very
melanomas
distinct
progression.
expression
and
in
functional
in melanoma
logical
the
could
investigated
cell
CD44v5
to facilitate
relevance
as malignant
isoforms,
of CD44v5
that
of isolated
melanoma
as well
of
and stimulate
cytokines
The
is being
the in
explanation
are known
the proliferation
provision
mel-
during
evidence
which
tissue.
growing
capacity
cells
These
lymphoid
factor
possible
is preliminary
lymphocytes.
the surrounding
ons
There
One
to antigen-presenting
to produce
support,
shown).
locally
expression
Cancer
29.
Wielenga.
van
den
V.
Berg,
J. M.,
F. M.,
variant
Ponta,
proteins
Cancer
Res.,
Heider,
K.
H.,
in human
53:
H.,
Offerhaus,
Herrlich,
colorectal
4754-4756,
P.,
and
J. A.,
Pals,
cancer
Adolf,
S. I.
is related
G.
R.,
Expression
to tumor
1993.
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer
Research.
456
CD44
30.
Variant
in Melanoma
Isoforms
Kaufmann,
M.,
Heider,
Ponta,
H., and Herrlich,
cancer
and
length
K.
H.,
P. CD44
of survival.
Sinn,
variant
Lancet,
3 1 . Liu, A. Y. Expression
Lett., 76: 63-69, 1994.
H.
345:
of CD44
P.,
von
Minckwitz,
exon epitopes
in
615-619,
G.,
cancer
cells.
Cancer
Guo, Y., Ma, J., Wang, J., Che, X., Nanila, J., Bigby, M., Wu, M., and
Sy, M-S. Inhibition
of human melanoma
growth and metastasis
in vivo
32.
by anti-CD44
33.
monoclonal
DalI,
Surface
P.,
Cancer
Res.,
Kaufmann,
M.,
Ponta,
of distinct
CD44
Res., 54: 3337-3341
variant
1994.
Hekele,
antibody.
A.,
expression
noma.
Cancer
34. lerpe,
GUnthert,
A., Franke,
U. Occurrence
and pathological
F.,
Stark,
of CD44
conditions.
,
54:
1561-1565,
H.,
epitopes
and
1994.
Herrlich,
on cervical
P.
carci-
Dtsch.
A. A., Smith,
Ges.
Pathol.,
1993.
77: 276-281,
lecular
and
University
of Tokyo
36.
Guo,
Y., Liu,
Potential
metastasis
422-426,
37.
G., Wang,
use of soluble
in patients
X., Jin,
D., Wu,
M.,
Ma,
B cells
J., and
CD44 in serum as indicator
of tumor
with gastric
or colon
cancer.
Cancer
Sy, M. S.
burden
Res.,
and
54:
Oberg,
K.
1994.
A., Gobl,
Different
splice
in other
981-986,
subtypes
1994.
variants
of
A., Eriksson,
of CD44
B., Skogseid,
are expressed
endocrine
pancreatic
B., and
in gastrmnomas
tumors.
Cancer
38. Shtivelman,
in neuroblastoma
E., and Bishop,
J. M. Expression
of CD44
cells. Mol. Cell. Biol.,
11: 5446-5453,
39. Kee,
Andrulis,
Dadi,
B. L.,
H.
J. Cell.
Iran-Paterson,
R., Quackenbush,
M. CDIO and CD44 genes ofleukemic
40.
M., Gron-Virta,
Salmi,
Physiol.,
no evidence
148:
H., and
S. Regulated
expression
of exon v6 containing
isoforms
of
CD44 in man: downregulation
during
malignant
transformation
of tumors of squamocellular
origin.
J. Cell Biol.,
122: 431-442,
1993.
Mackay,
C. R., Maddox,
Y. F., Wijifels,
G. L., Mackay,
I. R., and
Walker, I. D. Characterization
of a 95,000 molecule
on sheep leucocytes
homologous
to murine Pgp-1 and human CD44. Immunology,
65:
93-99.
42.
1988.
Halaban,
Smeets,
R.,
Ghosh,
S.,
Duray,
A. B. Human melanocytes
cultured
Dermatol.,
93: 95-101, 1986.
lines
R.,
derived
from
Noordmeer,
I. A.,
G. N. P., Jansen,
D. F. C. M.,
characterization
a series
of solid
P., Kirkwood,
J. M., and Lerner,
from nevi and melanomas.
J. Invest.
Beck,
J. Natl.
cells
KrUse-Wolters,
mice.
and Ruiter,
melanoma
Int.
xenografted
1991.
M.,
Ruiter,
D. J.,
expression
in
C. F. J., Cornelissen,
J. L. M.,
of a human
in nude
Histochem.
J., 20:
L. M. H. A.,
D. J. Establishment
cell line (MV3) which
48: 85-91 , 1991.
J. Cancer,
75-80,
and
is highly
1988.
P., and Sacchi,
N. Single-step
by acid guanidinium
thiocyanate-phenol-chloroform
Biochem.,
162: 156-159, 1987.
method
57. Southern,
E. M. Detection of specific sequences
ments separated
by gel electrophoresis.
J. Mol. Biol.,
D., and Matsumura,
Y. Deranged
J. Pathol., 171: 249-250,
1993.
the hyaluronic
acid cell
metastatic
dissemination.
60. Lesley,
P. W., and Hyman,
vation
binding
61.
J., Kincade,
gene
activity
in
receptor.
Its role in
Bull. Cancer
(Paris),
R. Antibody-induced
acti-
of the hyaluronan
receptor
function
of CD44 requires
multivalent
by antibody.
Eur. J. Immunol.,
23: 1902-1909,
1993.
Koretz,
K.,
M#{246}ller,P.,
Lehnert,
Herfarth,
C. Effect of CD44v6
Lancet, 345: 327-328,
1995.
62.
of RNA isoextraction.
among DNA frag98: 503-517,
1975.
CD44
59. Thomas,
L. CD44,
neoplastic
invasion
and
80: 833-844,
1993.
Seiter,
H., Herrlich,
Johnson,
J. P., Demmer-Diekmann,
M., Meo, T., Hadam, M. R.,
and Riethmtiller,
G. Surface antigens of human melanoma
cells defined
by monoclonal
antibodies
I. Biochemical
characterization
of two anti-
tumors.
55. Cattoretti, G., Berti, E., Schir#{243},
R., D’Amato, L., Valeggio, C., and
Rilke, F. Improved
avidin-biotin-peroxidase
complex
(ABC) staining.
malignancy.
R., Kalimo,
S. A., lodaro,
G. J., Arnstein,
P., Kersey,
W. P. In vitro cultivation
of human
tumors:
and non-metastasizing
human melanoma
Clin. & Exp. Metastasis,
9: 259-272,
54. VanMuijen,
58. latin,
Jalkanen,
41.
Versteeg,
Anal.
1991.
P., Grenman,
1985.
and Schrier,
P. I. C-myc downregulates
class
I HLA
human melanomas.
EMBO J., 7: 1023-1029,
1988.
lation
E. J.,
cells
cells
1979.
56. Chomczynski,
of transformation-related
414-420,
K., Sointu,
54:
is repressed
1991.
K.,
I. L., and Letarte,
and malignant
cell lines show
alterations.
but not
Res.,
Press,
of cell
metastatic
Chaudhry,
342-350,
melanoma
Cancer Inst., 51: 1417-1423,
1973.
52. VanMuijen,
G. N. P., Cornelissen,
L. M. A. H., Jansen, C. F. J., Figdor,
C. G., Johnson, J. P.. Brocker, E. B., and Ruiter, D. J. Antigen expression
53.
activated
1993.
15:
51. Giard, D. J., Aaronson,
J. H., Dosik, H., and Parks,
phomas:
characterization
and epithelial
malignancies.
with normal
3539-3547,
(Rockville),
K., Epstein,
of human
Dzarlieva, R. I., Breitkreuz, D., Hennes, B., Engstner,
and Fusenig, N. E. Heterogeneity
of human malignant
in vivo and in vitro: role of experimental
systems.
In: J.
Klaus, E. Paul, and M. Schartl (eds.), Biological,
MoClinical
Aspects
of Pigmentation,
pp. 435-447.
Tokyo:
of metastasizing
into nude mice.
comparison
Blood,
82:
A. J., Fukuyama,
properties
50. Tilgen, W.,
M., Matzku, S.,
melanoma
cells
Bagnara,
S. N.
35. Salles,
G., Zain,
M., Jiang,
W. M., Boussiotis,
V. A., and Shipp,
M. A. Alternatively
spliced CD44 transcripts
in diffuse large-cell lymand
H. S., Hackett,
S. H. Biological
In Vitro
establishment
H., Gustmann,
C., Mackay,
W., and
and its isoforms
under
orthological
Verh.
Creasey,
in culture.
1995.
in prostate
49.
W. L., and Madin,
breast
primary
S., Arch,
R., Reber,
P., Matzku,
I.,
Hinz,
on survival
S., Komitowski,
S., and Zoller,
U.,
Otto,
H.
in colorectal
F.,
and
carcinoma.
D., Hofmann,
M.,
Ponta,
M. Prevention
of tumor metastasis
177: 443-455,
1993.
43.
formation
gens found on cell lines and fresh
J. Immunol., 11: 825-831,
1981.
Eur.
63. ZOller, M. CD44
variant
expression,
lymphocyte
homing
and lymphogenic
metastasis.
In: W. Siess, R. Lorenz, and P. C. Weber (eds.),
Adhesion
Molecules
and Cell Signaling:
Biology
and Clinical
Applications, pp. 201-218.
New York: Raven
Press,
Ltd., 1995.
L. A., Oettgen, H. F., and Old,
malignant
melanoma:
mixed
64. Cooper, D. L., and Dougherty,
G. J. To metastasize
or not? Selection of CD44 splice
sites. Nat. Med.,
1: 635-637,
1995.
44. Carey, I. E., lakahashi,
L. J. Cell surface antigens
hemadsorption
melanoma
assays
cells.
Proc.
tumors
of diverse
I., Resnick,
of human
for
humoral
NatI.
Acad.
immunity
Sci.
USA,
to
73:
tissue
origin.
cultured
autologous
3278-3282,
D’Urso,
C. M., Wang,
Z., Cao, Y., Tatake,
R., Zeff, R. A., and
Ferrone,
S. Lack of HLA class I antigen
expression
by cultured
melanoma cells FO-l due to a defect in B2m gene expression.
J. Clin. Invest.,
87: 284-292,
1991.
46. Giacomini,
P., Imberti,
L., Aguzzi,
A., Fisher,
P. B., Trinchieri,
G.,
and Ferrone,
S. Immunochemical
analysis
of the modulation
of human
melanoma-associated
antigens
by DNA
recombinant
immune
interferon. J. Immunol.,
135: 2887-2894,
1985.
Bean,
M. A.,
Bloom,
B. R.,
Herberman,
R. B., Old,
L. J., Oettgen,
H. F., Klein, 0., and Terry, W. D. Cell-mediated
cytotoxicity
for bladder
carcinoma: evaluation of a workshop. Cancer Res., 35: 2902-2913,
1975.
48. Godal, A., Fodstad,
0., and Morgan,
lines showing
striking
inherent
differences
ins containing
holotoxins.
J. Natl. Cancer
A. C. Human
melanoma
CD44.
J. Exp.
cell
in sensitivity
to immunotoxInst., 77: 1247-1253,
1986.
Med.,
Arch, R., Wirth, K., Hofmann,
P., and Zoller, M. Participation
M., Ponta,
in normal
metastasis-inducing
257: 682-685,
of CD44.
65.
1976.
45.
47.
by anti-variant
splice
1992.
66. Z#{246}ller,
M. CD44:
evidence
425-438,
67.
Herrlich,
Seiter,
Zdller,
epidermal
physiological
for organ-specific
1996.
metastases
68.
variant
H., Matzku, S., Herrlich,
immune
responses
of a
Science
expression
metastasis
(Washington
of distinct
formation.
DC),
isoforms
J. Mol.
Med.,
as
73:
P., Z#{246}ller,
M., Pals, S. I., and Ponta, H. CD44 splice variants:
meet lymphocytes.
Immunol.
Today,
14: 395-399,
1993.
S.,
Herrmann,
M. Expression
tumours.
K.,
of splice
Virchow’s
M#{246}ller,P.,
Patzelt,
variant
of CD44
Archiv.
Pathol.,
E.,
Tilgen,
on human
in press,
W.,
and
skin and
1996.
69. Herold-Mende,
C., Seiter, S., Jourdan,
U., Herrmann,
K., Born,
A. I., Patzelt, E., Z#{246}ller,
J., Bosch, F. X., and Ztiller, M. Expression
of
splice
variants
of
carcinoma
of head
CD44
on squamous
and neck. J. Pathol.,
epithelia
in press,
and
1996.
squamous
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer
Research.
cell
Expression of CD44 variant isoforms in malignant melanoma.
S Seiter, D Schadendorf, K Herrmann, et al.
Clin Cancer Res 1996;2:447-456.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://clincancerres.aacrjournals.org/content/2/3/447
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer
Research.