Vol.
4, 791-797,
March
1998
Clinical
Overexpression
for
of Lerk-5IEplg5
Increased
Human
Tumorigenicity
Malignant
Thomas
Jung,
Kobayashi,
Michael
McClelland
Center.
Toronto,
Metastatic
Welsh,
Landthaler,
expression
possibly
of growth,
tumorigenicity,
melanomas.
and
San Diego. California
92121
of Dermatology,
University
[T. V.,
of
Ontario
M4N
3M5.
Canada
tyrosine
are
ligands
of eph-related
expanding
receptor
family
tyrosine
ki-
thought
to
of genes
play an important
role in the development
and oncogenesis
of various
tissues.
However,
very little experimental
evidence supports
this hypothesis.
Using RNA fingerprinting,
we detected
increased
melanocytes
as
expression
a
of Lerk-5
to the
response
sion
Therefore,
in various
in human
which
role of the Lerks
progression.
mRNA
tumor-promoting
12-O-tetradecanoylphorbol-13-acetate,
possible
in melanoma
we
studied
melanoma
cell
Lerk-5
lines
drug
suggests
a
tumorigenesis
and
and
mRNA
tissues
a 3.9-fold
compared
MelS).
to the
Progeny
mRNA
ent
mice
with
Lerk-5
or undetectable
nevi (n = 9; P
UVB
data,
expression
in advanced
mRNA
<
0.001).
(n
SK-
low
Lerk-5
increase
in
it was selected
for higher
resistance
by passaging
in
high-dose
metastases
very
=
irradiation.
we
22)
expression
We conclude
found
Consist.
high
primary
but
Received
6/9/97;
revised
12/29/97;
levels
of
lower
Dermatology,
94049
944
University
Regensburg.
9651;
E-mail:
of Regensburg,
Germany.
Phone:
[email protected].
944
9560;
Fax:
a potential
for new
experiments
not
sequence
known
to
these,
the expression
interest
because
a pivotal
tags
be
origin
lines
but
not
Lerk-
1 may
nomas
(8).
normal
a family
also
of ligands
be
a type
Lerk-2
that
have
and
12)
membrane-bound
can
induce
(10).
All
gesting
in malignant
mela-
is a member
(9,
protein
10).
that
Elk-L3fNLerk-2
At
is
(13).
requires
share
cell-to-cell
domains,
can become
strong
most
sequence
cells,
function
including
five
phosphorylated
in which
We
analyzed
to
these
( 14) and
elk,
and
htk
of
invariant
tyrosine
themselves,
sugeph-RTK
pathways
in
in addition,
their
of tyrosine
eytoplasmie
kinases
(e.g.
.
by
growth
factor
receptor
tyrosine
kinase),
allowwith other proteins
that may activate
signaling
in ligand-expressing
study
provides
role
and
as substrates
of
conservation
a dual role for this type of transmembrane
they can probably
activate
signaling
sible
seven
similar
contact
hek,
(5):
may
of
least
Binding
of the eph-RTKs
ligands
receptor-expressing
cell
that
share close amino
acid identities,
ranging
been described
(3, 1 1 ). Lerk-5
is predicted
ligands
their eytoplasmie
residues,
which
was
suggesting
as Ebf2/HTK-L,
phosphorybation
three
B6l
melanoma
factor
eph-RTKs
1 transmembrane
(4,
signal-
and, possibly,
in
in tissues
of neuro-
melanocytes,
referred
is of partie-
Lerk-l/protein
growth
of the
mRNA
of eck-expressing
be an autoerine
different
ligands
from 28 to 59%,
to
Recently,
cultured
known
melanocytes
of the eph-RTK/Lerk
ectoderma]
the growth
in
of Lerk5
role
was postulated
in differentiation
of various
tissues,
particularly
(3-7).
melaof more
and of several
expressed
ing system
oncogenesis
to stimulate
of human
of Lerk-5
the
Lerk-5
the
may
in melanoma
expression
cells (5).
first description
be involved
of a functional
and
tumorigenesis
patterns
of
suggests
and
Lerk-5
a pos-
progression.
mRNA
in a
to
and CA68822,
Nr. 97.021.1.
Department
of
Franz-Josef-Strauss-AlIce
49 941
solely
eph-related
system
(I) led to the detection
Among
pathways
This
12/29/97.
1734
expressed
were
platelet-derived
ing interactions
of this article were defrayed
in part by the
This article must therefore be hereby marked
advertisement
in accordance
with
18 U.S.C.
Section
indicate this fact.
I This
work was funded
in part by NIH Grants
NS33377
as well as by Wilhelm
Sander-Stiftung
(Munich)
Grant
2 To whom
requests for reprints should be addressed,
at
in human
as well as a target
scanning
ular
domains
in benign
melanocytic
that increased
Lerk-5
accepted
markers
(2).
context
The costs of publication
payment of page charges.
new
that
ligand
malignant
significantly
potential
abilities
signaling
the RAP-PCR3
Lerk-5fEplg5,
when
and
a 5-fold
experimental
and
abundance
an increased
metastatic
the yet to be elucidated
eDNA
a dozen
mRNAs
expres-
of melano-
(RPMI-7951
showed
expression
when
and multicytokine
mRNA
melanomas
lines
cell line with
(WM35)
or repeated
these
transcript
cell
of a melanoma
abundance
Lerk-5
mRNA
tumorigenicity
nude
increased
primary
or induces
and
for molecular
that used
shown
cytic tumors
by semiquantitative
reverse
transcriptionPCR. Modest
expression
of Lerk-5
mRNA
was found
in two
melanoma
cell lines derived
from early primary
tumors
(WM3S
and WM1645B);
two metastatic
cell lines tested
showed
in
kinase/Lerk
Systematic
than
a rapidly
Marker
INTRODUCTION
[R. S. K., H. K.]
ABSTRACT
The Lerks,
791
therapies.
nocytes
nases,
reflects
This makes
receptor
Germany
[T. V., W. S., M. LI: and
Research,
Sunnybrook
Health
Science
Biology
A Novel
Potential
new source
Sidney Kimmel Cancer Center.
J. W., B. J.. M. M.]; Department
Regensburg,
93049
Regensburg.
of Cancer
RNA:
Research
Melanomas1
Michael
Hiroaki
Division
and
Wilhelm
Stolz, John
Robert
S. Kerbel,
Vogt,2
Barbara
Messenger
Cancer
1 1,
49 941
3 The
abbreviations
used are: RAP-PCR,
RNA arbitrarily
primed
PCR;
eph-RTK,
eph-related
receptor tyrosine kinase; TPA, I 2-O-tetradecanoylphorbol- I 3-acetate:
SSCP, single-stranded
conformation
polymor-
phism;
RT-PCR,
reverse
transeription-PCR;
of Lerk-5
transcript;
PKC. protein
kinase
RALT,
relative
abundance
C.
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer
Research.
792
Lerk-5
mRNA
series
of melanoma
ditions
Expression
and
nant
lines
melanoma
under
are paralleled
molecule
experimental
Our
metastatie
data
and
we
could
diagnostic
the
1-3
4.6
B
7-9
mRNA
of
marker
can
location
of this
on
chromosome
tion
to
the
accession
I 3q33
current
no.
GenBank
AND
Culture
man
Caucasian
netics
Corp.
be
newborn
factor.
5 p.g/ml
.5
CA)
0.5 p.g/ml
and
tumonigenicity
nude
mice;
35-P2-N2,
respectively
lines from
lymph
HTB66/RPMI-795
cells
( 15).
Type
Culture
were
grown
analyzed
were
to RNA
similar
to the
the UVC
energy
fast
Fig.
growing
cell
by two
were
used
5%
at the
Regensburg
the
expert
a
with
collected
(Re-
patients
Diagnoses
M. L.).
All
(Life
supplemented
of
place.
MD).
was
of the
ob-
tissues
histopathologists
As a UV
customized
prior
source
for UV
apparatus
(Strat-
with a built-in
flux measurement
device.
by the manufacturer
had a continuous
observed
(<290
nm)
nm)
with
with
with
the
sun,
is negligible.
in the UVA
range
a peak
shorter
so
About
(320-400
at 312
is
the
emission
in
15%
ofthe
total
Mean
UVB
rates were 20 i/s.
RAP-PCR
and
Isolation
Transcripts.
Total
cellular
RNeasy
spin
the
column
cells
culture
purification
were
dish,
directly
whereas
Pieces
frozen
and
removal
Differentially
was extracted
kit
lysed
tissues
to homogenization.
after
of
RNA
(Qiagen,
Chatsworth,
of lysis
required
of
of the diagnoses
by expert
histopathologists
based
on sections
and routine
staining
parts
of
were
cut
sample.
sected
the
same
and
tumor,
stained
Subsequently,
using
10-p.m
to
thick
epidermis.
the
remaining
pieces
dermis,
were
the
sections
and
AP-l 1 (5’-AGGGGCACCA-3’)
The RAP-PCR
products
were
and
loaded
three
lanes
RNA
group
and were electrophoresed
the
were
to
frozen
I mm)
s.c.
into
fat
a cell
were
lysis
Chatsworth,
another
stained
nests.
gen,
and
The
homogenate
of
Lerk-5
AP-4 (5’-GCACCAGGGG-3’).
side by side so that each set of
on an 8
the
total
fingerprint,
synthesis
strand
one
dis-
amplified
The
(Qiagen.
and
of
six
RNA
was
treated
eDNA
The
fingerprints
arbitrary
RAP-PCR
primers.
products
fingerprint
with
DNase
performed
that
generated
for
Isolation
was
was
first
in Fig.
these
of putatively
achieved
as
the
isostrand
for
using
I as
produced
fragment
is shown
(Qia-
genomic
was
(5’-GCACCAGGGG-3’)
RNA
the desired
product
with native
SSCP
Cloning
and sequencing
of the selected
ard
RNA
primers
Lerk-5
to further
DNA
remaining
1” (5’-AGGGGCACCA-3’)
“AP-4”
Finally,
used
genomie
the
cellular
( 1 ). Arbitrary
the
pestels.
were
down
eliminate
of total
which
“AP-l
synthesis.
were
of
from
glass
from the remaining
tissue and
for correct
dissection
of the
columns
To
extracted
snap
were
with
taken
control
and to break
CA).
previously
bated,
was
Qia-shredder
described
is
homogenized
the bysates
Chatsworth,
various
removed.
CA)
10-p.m section
as an additional
skin
parts
buffer
up-regulation
melanocytes.
A. starved
normal
1-3) were treated with the tumor-
concentrations
of each
treatment
M urea/6%
polyacrylamide
gel.
Arrowhead,
band from which
we obtained
the Lerk-5
cDNA
fragment.
B, differential
expression
of the Lerk-5
mRNA
transcript
(arrowhead,
Lerk-5),
dependent
on TPA treatment,
was then confirmed
by relative.
semiquantitative
RT-PCR
using a piece of I 85 rRNA
as internal
standard (arrowhead,
S). Lane 1, starved normal
human
melanocytes:
Lanes
2 and 3, melanocytes
treated
with 32 nsi TPA for 4 and 24 h: Lane 4.
melanocytes
treated
with 32 nsi TPA for 4 h and simultaneously
growth
arrested
by a single
UVB treatment
(2000 J/m2). RT-PCR
products
were
eleetrophoresed
on a nondenaturing
(SSCP)
gel for 24 h. Therefore.
Lerk-5
mRNA
and internal
standard
are both represented
by two bands.
Quantitation
was performed
by [3-counting
of the upper
band,
corresponding
to Lerk-5
mRNA
abundance
relative
to the lower band of the
internal
185 rRNA
standard.
termed RALT.
the
Contaminating
three
prior
pieces
within
represented
(1). RAP-PCR
establishment
the
tumor
(up
binoculars.
collected
of
human
newborn
melanocytes
(Lanes
drug TPA (32 nM; Lanes
7-9) and with TPA in combination
with 0.02 mg/mI
cycloheximide
(Lanes
4-6).
Eight
h after the treatments, total RNA was prepared
and fingerprinted
at three 2-fold
dilutions (400. 200. and 100 ng of total RNA) using the arbitrary
primers
described
to
(W. S. and M. L.),
of paraffin-embedded
sections
localize
microscope
skin
at -80#{176}C.After
reveals
normal
promoting
DNA,
CA).
buffer
mierodisseetion
tumor-carrying
stored
Amplified
using
the
by addition
fingerprinting
human
tumor
flux
Cultured
RAP-PCR
in TPA-treated
nm.
wavelengths
nm).
I
mRNA
Mela-
formulation
of
consent
S. and
we
is emitted
TPA,
nM)
(Roekville,
NY)
took
(W.
pattern
range
(16
melanomas
were
were selected
for
1640
tumors
UVB light (290-320
of emission
energy
of
decrease
for-
fibroblast
extract.
two
University
confirmed
selection.
spectrum
The
Island,
operations
agene. San Diego.
CA)
The UV lamps provided
<S
Clo-
resistance
by pasto as 35-P2-Nl
and
Collection
Written
extraction
resistance
multicytokine
referred
Dermatology,
the
10 ng/ml
in a RPM!
melanocytic
before
basic
pituitary
In addition,
Grand
Germany).
from
in a MCDB153
human
<S
hu-
metastases
of malignant
melanomas,
HTB7O/SK-MEL-5,
were
purchased
Inc.,
of
grown
bovine
are
from
Department
and
and
these
FCS.
tamed
GenBank
purchased
from early primary
and some WM35
cells
Technologies.
Tissues
were
1 ng/ml
p.g/ml
node
1 and
American
gensburg,
(new
Cryopreserved
hydrocortisone.
15
enhanced
sage in
melanoma
informa-
<
Samples.
with
cell lines derived
and WM1341B,
from
sequence
entry
mebanocytes
Diego.
insulin.
noma
WM35
database
Tissue
supplemented
growth
further
METHODS
and
(San
mulation,
add
[.5
U81262).
MATERIALS
Cell
and
<
-
this
<
gene
4
malig-
applications
confirm
A
that
of
of Lerk-5
clinical
as a prognostic
con-
indicate
potential
by up-regulation
potential
Furthermore,
various
tumors.
and
Therefore,
envisioned.
Melanomas
of melanocytie
dedifferentiation
expression.
novel
cell
tissues
increasing
in Malignant
second
This
1A.
RNAs
and
differentially
by purification
gels as described
eDNAs
followed
procedures.
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer
Research.
of
(16).
stand-
Clinical
Confirmation
quantitative,
tissue
of Differential
Relative
samples
RT-PCR.
with
low
tive,
relative
RT-PCR
TX).
The
QuantumRNA
paring
relative
fication
invariant
still
in
were
fication
mRNA
adjusted
transcript
of
only
are
these
transcript
that
(Competimens),
18S
both
The
single
tube.
For
Lerk-5
which
produce
Lerk-5
mRNA.
mega,
Madison,
10-fold
2X
standard
amounts
(Perkin
0.4
each
mixture,
and
185
loaded
onto
maximum
the
2X
pJ) were
PCR
primer
analysis
RALT
was
Lerk-5
the
185 RNA
Differences
canee
6%
(SSCP
gel).
+
0.55
+ +
mixture
cycled
with
20
ib of
polyacrylamide
of the
to
the
Lerk
software
nucleotides)
product
and
and
(Ambis
the
San
internal
data
Diego,
of the counts
(488 nueleotides),
different
tissues
for
per-
subsequent
Inc.,
and the lower
the Mann-Whitney
gel
was
EDTA
buffer.
Gels
paper and dried under
3-eounting
CA).
ofthe
as shown
were tested
and
1 157,
addition,
internal
in Fig. lB.
for signifi-
pared
from
starvation
Transcript.
normal
human
melanocytes
were surveyed
by RAP-PCR.
from
fingerprint
of the 3’ Untranslated
mRNA
The
fingerprint
this
fragment
which
the Lerk-5
indicated
in the
a >4-fold
TPA-treated
group.
Database
analysis
performed
on-line
using
Biotechnology
gov/).
The
Information
sequence
was
Region
Total RNAs
fragment
increased
group
versus
was
isolated.
amplification
the TPA-starved
of the sequence
of this fragment
blastnlnr
at the National
Center
web
found
site
pre-
treated
with TPA after
Fig. 1A shows the RNA
eDNA
of
was
for
(http://www.nebi.nlm.nih.
to be
100%
homologous.
24 h
++
+++++
0.48
as the ratio of the counts
no.
is known
the
to map
+
of the Lerk-5
in
no. HSU16797),
mRNA
sion
no.
some
HUMHTK),
mRNA
HTK
13q33,
clone
eDNA
kinase
which
which
sequence
sequence,
Lerk-5
mRNA
(new GenBank
Lerk-5
previously
transcript
accession
mRNA
accession
overlap
with
gene
(GenBank
to Lerk-5
gene
anticipated
of
modulated
by TPA.
quiescent
features,
to study
senescent
and
limited
is commonly
(20,
each
TPA-free
which
21).
day
In this
during
medium
by
status
otherwise
the
1-6.
On
of
of
cycling.
generations
become
cells
day
20
of
of culture
quickly
experiment.
of
adoption
as a supplement
a period
culture
abun-
growth
15-20
bases
Melano-
A tissue
transcript
rapid
days
for
of 2902
an activated
to about
used
melanocytes,
die
medium
received
induces
Lerk-5
of the
Human
characterized
importantly,
remains
TPA
medium
fresh
most
growth
(17-19).
TPA
melanocytes,
interspecific
HUMCHI3GEN
to a total
in Normal
we
on chromo-
by
the
cytes Reflects
Growth
Stimulation
by TPA.
experiment
was designed
to investigate
Lerk-5
dance
aecesThus,
entry for the human
sequence
information
was expanded
no. U8l262).
Expression
no.
the human
(10).
Lerk-5
fusion
with the previous
the available
(GenBank
transmembrane
(GenBank
ligand
By
In
clone has a 294-base
overlap
mRNA
(GenBank
accession
is identical
(10).
1007
l3q33.
in mouse
of the human
was
analysis
bases
3’ untranslated
hepatoma
as a 435-base
the location
back-cross
the
I 3GEN
Lerk-5
transmembnane
to
sequence
mouse,
as well
between
to chromosome
homology
168 19) and
MUSHTK).
The HUMCH
with
the entry
for human
h,
received
7,
all cells
a time
that
induces
G0 in melanocytes
(19, 21). On day 8, the cells got fresh
medium,
either
supplemented
with TPA (32 nM) or not. The
TPA dose used was within a broad, dose range (10-100
nM) that
uniformly
activates
stimulating
different
lar location
to
MMU
ligand
HUMCH13GEN
significant
Lerk-5
no.
kinase
cells
of Parts
2.11
which
of
normal
U test.
RESULTS
Lerk-5
0.68
it showed
However,
Characterization
mice
++
+++
UVB
accession
tumor-like
of the Human
in nude
+ +
+++
TPA,4h
was defined
GenBank
of the upper
band
4 h
and 18S rRNA
internal
standard
PCR product
on the gels.
‘, Relative
regulation
in comparison
to quiescent
normal
melanocyte: +.0.00-0.49:
++.0.50-0.99:
+++.
1.00-1.49:
++++.
1.501.99; +++++.
>2.00.
accession
pM
through
Three
mm).
“ RALT
PCR product
regions
0.75
Eleetrophoresis
as the ratio
standard
between
using
0.11
a nondenaturing
Ambis
(510
WM35
can confirm
calculated
band
1.44
1.19
hepatoma
subjected
using
HTB66
HTB7O
were mixed
with 1 2 p.1 of formamide
dye
at 95#{176}C
for 2 mm.
Two ib of this solution
at 80#{176}C.Bands
were
to 10 p.1 of
II, 4 mrvi MgCl2,
primer
72#{176}C,1
The
Polymerase
[a-32PIdCTP,
formed
for 24 h at 5 W in 0.6X Tris-borate
were then transferred
to 3-mm Whatman
standard
added
and
WM1645B
of
(Pro-
RNA.
DNA
buffer
at 8:2]
5;
sequence
of total
1 .6 i.l of standard
resolution
vacuum
coding
the RT-System
AmpliTaq
s; 66#{176}C,30
complete
reaction
buffer and denatured
were
and
used,
p.Ci/reaetion
nRNA
(94#{176}C, 30
5’-
AC-3’
were
I pg
(1.5
CT),
2
primers
by
with
solutions
dNTP,
Competimer:
cycles
starting
Norwalk,
the
from
++++
+++++
TPA,
AGA
TGC
ACC AGC-3’
produced
[2 units/reaction
Elmer,
primer
were
eDNA
mixture
mi
product
eDNAs
WI),
CAC
ACC
+
1.99
was
The
in a
pattern”
0. 19
Passaged
are modi-
that
RALT”
2.26
0.62
0.56
of competitor
primers
human
TPA.24h
TPA/UVB.
of the target
amplification,
AGA
AGG
a 5 lO-bp
diluted
PCR
eDNA
AGC
AAG
is
ampli-
fled at their 3’ ends to block extension
by DNA polymerase.
cycling
of both the target and the standard
was performed
TAA
AGA
CCA
5’-TAC
Tl’C AGC
in normal
cell lines
TPA.4h
the
PCR
for the fragment
b8S RNA
rRNA
expression
melanoma
No TPA
an
experiments
of the amplification
i.e.,
as
if
amplified.
of increasing
Lerk-5
mRNA
and malignant
Treatment
Normal
melanocytes
coampli-
while
Pilot
of the eoampbifying
addition
possible
was
Differential
melanocytes
793
Research
Regulative
for eom-
rRNA
analyzed
conditions
I
Cell line
Austin,
by
1 85
amplification.
to the efficiency
by the
primers
is
reference
phase
efficiency
then
This
the
to determine
Lerk-5
standardized
of
Table
semiquantita-
Ambion,
a method
fragment
standard.
exponential
of the
we used
provides
abundances
and
performed
of small
module;
conserved
internal
by Semi-
to the analysis
of RNA,
module
transcript
transcript
Expression
Due
(QuantumRNA
of a highly
target
yield
Gene
Cancer
arrested
growth
PKC
of human
isoforms
( I 9, 2 1 ). Furthermore,
by
irradiation
with
melanocytes,
and
a fraction
a previously
probably
modulating
their
of
determined
cells
by
cellu-
was
dose
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer
Research.
of
794
Lerk-5
mRNA
Expression
in Malignant
A
Melanomas
B
C
Malignant
1
2
3
-
PLtr
on...
SI,
;‘.lJP
4
..
#{149}
6
7
8
9
-
“#{149}
c.w a..
-#{149}$
%13m
1
.
5
SoS
-on
#{176}
-
“i
Melanomas
10
11
_
.
i*
-..pswv
.ka,tr
7
8
.“4,
#{248}n.
D
Melanoma
Metastases
‘-‘-ta’
,.,,
Melanocytic
E
_.
_____
on
Nevi
9
10
4
*II;=
Fig. 2 Relative semiquantitative
RT-PCR confirms Lerk-5 mRNA expression
as a putative indicator of higher tumorigenicity
and metastatie
ability.
A, Lerk-5
mRNA
expression
in human melanoma
cell lines. Lanes I and 2. cell lines derived from initial malignant
melanomas
(WM1645B
and
WM35); Lanes 3 and 4, metastatic
cell lines (RPMI-795l
and SK-Me15).
B, increased
expression
of Lerk-5
mRNA
in WM35
cells (Lane 1) selected
for higher tumorigenicity
by passage in nude mice (Lane 2) or selected
for UVB damage
resistance
(Lane 3). Short-time
treatment
with TPA (Lane
4, 4 h) also increased
Lerk-5
mRNA
abundance,
whereas
long-time
treatment
(Lane 5, 24 h) led to minor up-regulation
in this cell line. Up-regulation
of Lerk-5 mRNA abundance
in advanced malignant
melanomas
(C) and melanoma
metastases
(D) in comparison
to melanocytic
nevi (E; Lane 5 was
skipped).
Quantitation
was performed
by 3-counting
of the upper band corresponding
to Lerk-5
mRNA
abundance
relative
to the lower
band of the
internal
18S rRNA
UVB
(2000
Cells
from
ment;
24
TPA
and
subsequently
J/m2)
(RALT
as
predicted
1.99),
a similarly
observed
after
RALT
24 h ofTPA
showed
only
treated
with TPA and
irradiation,
suggesting
dance
and
of
Melanoma
Cell
Lines.
dance
studied
treat-
group
after
after
long-term
70% confluence.
Constitutive
mRNA
was found
in two
from
early
RALT
rapidly
starvation
was 0. 19. A 10-fold
were exposed
to TPA for 4 h
tested
high
Me15;
transcript
(RALT
(Fig.
mRNA
malignant
abundance
=
increase
2.26).
when
arrested
of Lerk-5
mRNA
Table
in
Cultured
was
However,
cells
in
1B).
Lerk-5
RALT
were
by UVB
abun-
melanoma
line
showed
compared
(WM35
and
lowest
Lerk-5
mRNA
mean
the
(RALT
(data
a 4-fold
with
=
not
increase
primary
RALT
1 .32).
progeny
were
produced
is known
that
to be highly
shown)
of
at
lines
The
cell
RALT
with
ratios
two
lines
when
1 and
SK-
low
con-
very
in nude
i.e.,
The
cell
is derived
subbines
passaged
tumorigenie,
was
14).
(RPMI-795
line
mean
expression
metastatic
the
cell
were
phase
WM1645;
0. 1 1 ; Fig.
stitutive
Lerk-5
mRNA
expression,
WM35,
early (thin) primary
melanoma
(15). Variant
parentals
growth
endogenous
expression
of
melanoma
cell lines derived
tumors
WM35
growing
in exponential
from an
of these
mice.
The
tumorigenie
in
of Matrigel,
and multicytokine
cells, a 5-fold
increase
of Lerk-5
was
found
(RALT
=
0.55).
Be-
abun-
cause
known
lines.
ness
Malignant
cell
The
cells
nude mice without
coinjeetion
resistant
(15). In the passaged
mRNA
transcript
abundance
1 summarizes
transcript
from
primary
0.33).
=
more
The
of the
harvested
in
RAP-PCR.
Endogenous
in various
were
about
Lerk-5
in the cell
treatment
Lerk-5
RNAs
found
a moderate
growth
the results.
Expression
was
abundances
simultaneously
a correlation
melanocytie
32 nsi TPA.
4 h after
in the TPA-treated
by
20 h of TPA
in cells that
and
with
harvested
to more immediate
changes.
we confirmed
TPA inducibility
cells after
was found
=
treated
were
transcript
compare
RT-PCR,
transcript,
RALT.
was done
how
treatment
By relative
quiescent
increase
groups
harvest
determine
Lerk-5
the
termed
all treatment
a second
h to
standard,
UVB
irradiation
of human
malignant
is also
melanomas
to increase
the aggressive-
for growth
and
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer
Research.
metastasis
Clinical
Table
Lerk-5
2
expression
in human
melanocytic
tumors
Lerk-5
Patient
initials
Histopathology
RT
LP
PT
AA
LE
BG
GC
MP
AF
LL
TA
ST
GS
EA
KF
MK
BC
SA
Melanocytie
Melanocytic
nevus
nevus
Melanocytic
Melanoeytic
Melanocytie
nevus
nevus
nevus
Melanocytic
Congenital
nevus
melanocytic
Congenital
melanocytic
nevus
Congenital
melanocytic
nevus
Nodular
Nodular
Nodular
melanoma
melanoma
melanoma
Lentigo
maligna
Nodular
melanoma
not correlate
expression
(li!:i!)
nevus
melanoma
spreading
with
Tumor
common
prognostic
Clark
thickness
0.02
0.02
0.02
0.03
0.03
0.28
0.33
0.53
0.59
0.28
0.55
0.56
0.62
0.65
0.70
0.77
0.82
I.01
Acrolentiginous
melanoma
Secondary
nodular melanoma
Secondary nodular
melanoma
Superficial
does
Cancer
Research
795
features
level
Prognostic
index”
1.50
4.80
III
V
5
78
1.90
IV
21
5.00
V
6.00
V
3.60
0.70
1.80
1.50
IV
II
III
IV
10
60
22
<1
2
ND”
2.30
3.10
IV
V
12
54
melanoma
GF
KH
SH
LH
LA
FD
LK
Wo
Nodular melanoma
Nodular melanoma
Melanoma
metastasis
Melanoma
metastasis
Melanoma
metastasis
Melanoma
metastasis
Melanoma
metastasis
Melanoma
metastasis
SN
Melanoma
metastasis
SD
TR
MH
VB
Melanoma
Melanoma
Melanoma
Melanoma
metastasis
metastasis
metastasis
metastasis
1
Prognostic
“
ND,
index
not
tumor
This
procedure
creased
70%
doses
repeated
and
4500
of 3500
and
parentals
formed
that survived
more
highly
Lerk-5
increase
autoradiography
marizes
the results
(RALT
metastatic
repeatedly
they
In
ni
of the corresponding
culture
TPA.
These
(19,
and
are
subsequent
Fig.
2B,
in
SSCP
gel.
Table
experiments.
inhi-
shows
nocytic
tumor
the
1 sum-
Was
to prepare
The
and
with
mRNA
± 0.05
levels
(0.82
Lerk-5
expression
cell
± 0.40
±
and
0.72
to the
SD)
but
we
found
melanocytic
significantly
melanomas
± 0.16,
eval-
we sought
to
by relative
RT-
experiments,
malignant
for nevi versus
melanomas
and versus
In adjacent
skin (dermis
and epidermis),
from
due
for pathological
Therefore,
in tissues
in the common
RALT
advanced
mela-
tissues
is limited
in tow
culture
fae-
in human
of sufficient
expression
added
fibroblast/growth
mebanomas
almost
our
Increased
in Comparthe caveats
of
of artificially
basic
prognosis.
expression
(mean
in
the presence
availability
the tissue
Consistent
Significantly
Their
Metastases
Bearing
in mind
serum,
primary
diagnosis
and
Lerk-5
mRNA
low Lenk-5
0.06
,
as calf
we assessed
nevi
uation
of
determine
e.g.
such
tissues.
melanocytic
metastases
moderate
20).
insulin.
creased
2. 1 1),
data
ton, and
nevi,
mRNA
=
Expression
experiments,
factors
very
to nontransLerk-5
culture
PCR.
we exposed
response
of malignant
mode of initial stimu-
capability
observed
of the cell
0.28).
mice
phenotype
32
mRNA
growth
need
corre-
nude
h. Similar
=
inthat
reached
the
positive
of the relative
with the concept
of a biphasic
cell lines to TPA. A response
and
mRNA
cell
h
doses
found
after 4 h (RALT
was associated
with only
accordance
melanoma
of growth
until
contained
24
of Lerk-5
has been
with
The progeny
experiment,
that
4 and
for >72
tumonigenic
expression
lation
times,
mRNA
medium
after
Lerk-5
in Malignant
Melanomas
and
ison with Melanocytic
Nevi.
subeultured.
harvested.
In a further
abundance
was
the 24-h exposure
bition
were
regrown
were
to the
to
a 5.3-fold
expression
(22),
with sublethal
clones
were
they
0.68).
=
harvested
cells,
transcript
whereas
J/m2
a stable
(RALT
were
counts.
to 3500
/m2 when they
point when DNA synthe-
Cells
two
then
of WM35
RNAs
by mitotic
J/m2, respectively.
similar
of WM35,
found
exposed
at a time
progeny
was
RNAs,
progeny
were
i.e.,
multiple
confluence,
sponding
was
and
to 4000
tolerated
multiplied
in long-term
gene
WM35
repeatedly
due to confluence.
is declining
isolated,
0.66
0.70
0.72
0.80
0.88
0.96
0.98
thickness
changes
cell line
doses of UVB. Parentals
reached
full confluence,
were
0.64
determined.
due to fundamental
we irradiated
the
sis
1.44
1.67
0.48
0.55
0.56
respectively;
metastases,
the RALT
in-
and
their
U test
P < 0.001).
values
were
between
0.2 and 0.25, which argues
for correct
mierodisseetion
of the common
nevi. Figs. 2, C-E, shows the autoradiographs
the corresponding
Higher
amounts
barge congenital
biology
tion
was
SSCP
Table
2 gives
of Lerk-5
mRNA
were also
nevi analyzed,
which
may
of congenital
found
gels.
between
nevi.
In primary
RALT
values
the detailed
of
results.
measurable
in the
indicate
a special
melanomas.
no correla-
and
prognostic
classical
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1998 American Association for Cancer
Research.
796
Lerk-5
mRNA
features,
Expression
such
prognostic
as tumor
index.
tissues
in Malignant
Melanomas
thickness,
Table
Clark
2 summarizes
of melanocytie
level
of invasion,
the results
or
obtained
vation
(causing
tumor-like
from
tumors.
most
direct
growth
and
and
neoplastie
DISCUSSION
cytie
Here,
we provide
bevel of expression
with
growth,
of Lerk-5
lines
tested,
(a)
mRNA
most
We
found
(15)
mRNA
was
a consistent
melanoma
nevi.
finding
metastases
The
was
relative
in large
congenital
of
pression
this
has
tumor
the
nevi,
assessment
of
ular marker
mebanomas.
The
with
and
extensive
the
ing the specifies
task
as Lerk-
Lerk-5
hek,
elk,
demonstration
mRNA
melanomas
of both
signal
transduction
of a series
htk
eek,
(10)
biological
Lerk-2
have
not
effects
yet
led
in response
a
to a clear
among
neuroectodermal
cells
(5).
Lerk-l/B6l
is expressed
in melanoma
Lerk-3IEHK-L/EFL-2
is exclusively
nervous
system
Lerk-5
as an indicator
potential
and the skin
Interestingly,
mRNA
imply
in
expression
melanocytes
our
and
culture
metastatic
functions
particularly
in
of the
experiments,
consistently
be
derived
from
cells
of
system.
cell
could
and
important
of the skin,
crest-derived
that
as our observations
tumorigenicity
mebanomas,
neural
observations
cell lines (8) and that
expressed
in the central
as well
and oncogenesis
melanocytie,
both
of high
in malignant
the development
(14),
The
induced
early
TPA
in
nonmetastatic
as TPA, have bong been
drugs (23). In cell eul-
tune,
it has
induces
the
malignant
cells
of
take
their
recognized
phenotype
on signs
malignant
that
in benign
TPA
cells
of changed
differentiation
counterparts,
which
(19).
a phenocopy
As
cytes
by
(20,
tumor-like
2 1 ). Therefore,
growth
as our
and growth
is paralleled
in cultured
study
of
a result,
by
mental changes
in gene transcription.
Accordingly,
TPA
the transition
from a quiescent
phenotype
to a phenotype
aetenized
overexpression
the tumors
or induces
metastatie
This
the
yet
makes
tis-
phenomena
and
system
mel-
(autocnine
eph-RTK-expressing
reflects
a potential
to
in
ma-
an increased
abilities
be
new
as well
a
and, prob-
in malignant
functional
markers,
for
pathways.
tumorigenicity,
prognostic
tyrosine
evidence
signaling
and
eph-
due to receptor-
indirect
within
cells
Because
of its cytoplasmie
of eph-RTKs
common
signaling
and
first
either
PKC
of major
factor-receptor
mRNA
It possibly
melanomas.
agnostic
therapies.
in
elucidated
source
of di-
as a target
for
new
newborn
demonstrates,
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such
known
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tumor-promoting
been
major
to
as
to receptor
activation.
However,
recent data concerning
the significance
of
Lerks for brain development
and neuronab
pathflnding
indicate
a crucial
importance
of this system
regarding
cell-to-cell
interaction
these
tumor
very
of growth,
eph-RTK/Lerk
( 1 1 ),and
the
metastases,
melanomas.
malignant
of tyrosines
activation
probably
pathway.
signals
growth
gives
due
identified
Because
ligand
to transduee
that Lerk-5
or between
are
lignant
ligands
elk
between
and their
potential
pathways
for
study
the consecutive
sues,
defin-
of eph-RTK
cross-talk
pathway)
activating
phosphorylation
this
anomas
with
able
of AP- I
(30), our data are congene is most probably
via the PKC
an
by platelet-derived
We conclude
ably,
and their
make
after
(e.g.,
vivid
I malignant
RTKs
and is probably
been
(27-29).
for TPA
the Lerk-5
transduced
stress-
changes
expression
has
promotion
represents
kinase-RTK),
molec-
interactions
by signals
with
of transformation
which
of tumor
via the PKC
resulting
enhanced
for
melano-
diacylglycerol
integrates
chance
hypermutability,
acts
The
the
important
in the
TPA
(26).
include
the
be
ligand
finally
sites
increase
ipso
also
that
offer
in melanocytie
promotion
confirmed
cellular
receptor
the concept
that
eo
may
(causing
may
involved
tumor
pathway
substrate
major
with
to TPA
the physiological
expression
driven
domain
common
which
at AP-l
and
like activity
In addition,
in stage
gene
RTKs
to genes
and
TPA
regarding
Lerk-5
marker
is a candidate
The
pathways
controlled
overex-
and
25).
reexposure
in differentiation)
access
It has been
induced
is the
sistent
tumors
easiest
by mimicking
the cellular
a special
molecular
to the eph-RTKs
receptor
and
of the
Lerk-5
as a new
pattern
discovery
the
benign
indicate
of ligand-receptor
of the involved
for
the
system.
oneogene
also
and
changes
differentiation,
components
Lenk-5
was
in histopathobogy.
of Lerks
11B61
for
used
expression
(9). The
in
may
significance
complexity
cross-binding
difficult
within
expression
prognostic
widespread
ligands
such
mRNA
increase
which
problem
Lerk-5
cell
metastatie
overexpression
of malignant
is a frequent
growing
5-fold
Therefore,
to be
diagnosis
which
overexpnesmelanoma
and
transformation
pathway
(24,
in
melanomas
of the skin and
to common
melanocytic
nevi,
potential
for differential
a
expression
entity.
correlates
ability
eonstitutive
malignant
mRNA
in malignant
as compared
highest
found
biology
by
Lerk-5
(c)
gene
the
of melanoma
cells for higher
resistance
by passage
in nude
accompanied
expression.
that
metastatie
in the rapidly
ones.
(b) In addition,
selection
tumonigenicity
and mubticytokine
mice
and
in all of the four
prominently
evidence
of the Lerk-5
tumorigenicity,
melanomas.
sion
experimental
of the transcript
increased
malignant
the first
quiescence)
growth
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R. Tyrosine
for eph receptors.
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elk:
10:
Molecular
expressed
USA, 89:
phosphoryla-
(Washington
1997.
6. Hirai, H., Maru, Y., Hagiwara,
K., Nishida,
J., and Takaku,
F. A
novel putative tyrosine kinase receptor encoded by the eph gene. Sdenee (Washington
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1987.
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Overexpression of Lerk-5/Eplg5 messenger RNA: a novel
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Clin Cancer Res 1998;4:791-797.
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