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Int. J. Cancer: 60, 73-81 (1995)
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
© 1995 Wiley-Liss, Inc.
LOW-METASTATIC HUMAN
MELANOMA
Marian A .J, W e t e r m a n 1*3, Nasser A ju b i1, Irma M .R. v a n D i n t e r 1, Winfried G .J . D e g e n 1, G o o s N.P. v a n M u i j e n 2,
Dirk J. R u i t e r 2 and Henri P J . B lo e m e r s 1
Departments of {Biochemistiy and 2Pathology, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
From a subtractive cDNA library» we isolated several cDNA
clones which showed differential expression between highly and
lowly metastatic human melanoma cell lines. One clone, desig
nated nmb, showed preferential expression in the low-metastatic cell lines and was chosen for further characterization.
Sequence analysis revealed that this clone represents a novel
gene, encoding a putative transmembrane glycoprotein which
showed the highest homology to the precursor of pMELI7, a
melanocyte-specific protein. nmf> RNA expression was absent in
most tumor-cell lines tested and not restricted to the melanocytic lineage. Transfection of a partial nmb cDNA into a highly
metastatic melanoma cell line (BLM) resulted, in 2 of 3 transfectants, in slower subcutaneous tumor growth and, in I of 3
transfectants, in reduction of the potential for spontaneous
metastasis in nude mice.
© 1995 Wiley-Liss, Inc.
Melanocytic tumor progression is thought to evolve through
several distinct stages, from normal melanocytes to highly
invasive melanomas capable of metastasis (Clark et al., 1984;
Hcrlyn et al, 1987). Monoclonal antibodies have been pre
pared recognizing antigens with a preferential expression in
one or a few stages of this process. Many of these molecules
have been cloned and mainly represent adhesion molecules or
growth-factor receptors (van Muijen et a lt 1990; Lehmann et
a l , 1989; Johnson et a l , 1989). Another approach, using the
differential and subtraction hybridization techniques, led to
the isolation of calcyclin (Wcterman etal, 1992) and thymosin
(3-10 (Wcterman et a lf 1993/;) as potential progression markers
for cutaneous melanoma. From a clinical point of view, such
markers as indicators of metastatic potential would be very
useful.
Although many markers have been isolated so far, it is useful
to obtain a large panel of such markers, since a single marker
or a few markers often show overlapping expression between
benign and malignant stages. Many proteins that are uprcgulated during progression arc also important in the normal
physiology of the cell. Thus, discrimination can only be
achieved when a large panel of markers is used. The study of
single markers can still be veiy useful, since it can provide us
with a better insight in the pathogenesis of tumor progression.
cD N A clones, which are expressed only in non-metastic or
low-metastatic cell lines or tumors, are candidates as tumorsuppressor genes. In the field of melanoma research, nm23 was
isolated upon comparison of highly and lowly metastatic
murine melanoma cell lines (Steeg et al, 1988). Although
expression in ncvoccllular nevi was lower than in melanomas,
/*w23 expression in melanomas, curiously, showed an inverse
correlation with disease progression (Florencs et al, 1992).
p53, another potential tumor-suppressor gene, was origi
nally reported to be mutated in a very large percentage of
melanoma lesions (Stretch et alf 1991). However, later reports
showed mutations in p53 only in a small percentage of
melanoma cell lines (Akslcn and Morkve, 1992; Luca et aI,
1993; Weiss et al, 1993). Another recently described gene,
designated p i6, is frequently deleted or rearranged in mela
noma cell lines (Kamb et al, 1994; Nobori et al., 1994). This
gene encodes a cyclin-depcndent kinase-4 inhibitor which
makes it a good candidate as a tumor-suppressor gene. The
exact extent and timing of these genes has yet to be deter
mined.
In an attempt to describe a subtraction library, we isolated
several differentially expressed cDNAs, 2 of which are ex
pressed only in low-metastatic cell lines and corresponding
xenografts and appear to represent novel genes. In this study,
we describe the characterization of one of them.
MATERIAL AND METHODS
Cell lines and xenografts
Hum an melanoma cell lines 1F6, 530, M14, McI57, B L M
(van M u ije n ^ a/., 1991«), M V3 (van M uijen et a l , 1991b), and
MV1 (van Muijen et al, 1991c) were cultured as described
before (Weterman et a1, 1993a). MV1 and MV3 were derived
from the same melanoma lesion after 1 and 3 passages in nude
mice, respectively (van Muijen et a1, 1991c). The MV1 cells
used for these experiments represent the lower metastatic
phenotype, giving rise to spontaneous métastases in about 10%
of the tumor-bearing animals. MV3 cells produce spontaneous
métastasés in approximately 90% of the tumor-bearing ani
mals. In this panel of cell lines, 1F6, 530, M.14 and McI57 are
low-metastasizing cell lines, whereas B L M and MV3 represent
the highly metastatic phenotype (van Muijen et al, 1991«).
Approximately 3 x ID6 cells were used for s.c. inoculation into
nude mice (nu/nu BALB/c; Bomholtgaard, Ry, Denmark).
A Northern blot containing total R N A of various cell lines
was generously provided by Dr, P, Quax (Gaubius Institute
IV V O , TNO, Leiden, The Netherlands). It contained R N A
from: Bowes1melanoma, HT1080 fibrosarcoma, Hela N IB SC
and Hela S3 cervix carcinoma, M CF7 mammary carcinoma,
A431 epidermoid carcinoma, Colo2Q5, CaCo2, HT29, SW620
and SW480 colon carcinoma, K ato III and I-IGT-1 gastric
carcinoma, HepG2 hepatoma, K562, U937 D K and I-IL60
myeloid leukemia cell lines, and HS766T pancreas carcinoma
cell lines. Human renal carcinoma cell lines, generously
provided by Dr. J. Rom ijn (Rotterdam, The Netherlands),
were all derived from the same patient (Weterman et al,
1993b). Rat organs that were used for a Northern blot were:
spleen, brain, liver, heart, muscle, kidney, testes, thymus,
prostate, lung, bladder, intestines and stomach.
Human tissues
After excision, large sections of melanoma métastases were
immediately frozen in liquid nitrogen and stored at -80°C.
Melanoma métastasés were processed individually and were
taken from patients other than those from whom nevi were
removed. For R N A isolations from common ncvoccllular nevi,
dysplastic (atypical) or congenital nevi, a representative slice
was taken. Most of the skin surrounding these lesions was
removed before the material was frozen in liquid nitrogen. The
remainder was processed for conventional histopathology. Dr.
R. Koopman generously collaborated in obtaining fresh hu
man material. It is guaranteed that this procedure did not
3To whom correspondence and reprint requests should be sent. Fax:
31 80540525.
Received: May 11, 1994 and in revised form September 4, 1994.
WETERMA.N ETAL.
74
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figure 1 - Northern-blot analysis of human melanoma cell lines
and xenografts. As a molecular-weight marker \ DNA restricted
with Hindi!I was used; nmb was used as a probe. Lanes A, 1F6; B,
530; C3 M l4; D, Mel57; E, M V I; F, MV3 and G, BLM RNA
isolated from cell lines; lanes H, 1F6; I, M l4; J, MV1; K, MV3 and.
L, BLM RNA isolated from xenografts. As a control a ribosomal
hybridization is shown.
hamper diagnosis. When nevi or normal skin tissue samples
were used, 6 to 22 lesions from 6 to 17 patients were pooled in
order to obtain enough m aterial A Northern blot containing
smooth-muscle-denved tumors (3 leiomyomas and 7 leiomyo
sarcomas) and normal uterus ( 4 x ) and adjacent tissue (4x)
was generously provided by Dr. T. Glouclemans (Utrecht, The
Netherlands).
RNA isolation and Northern-blot analysis
Total R N A was isolated as described by Auifray and
Rouge on (1980). 01'igo-deo.xy thymidine selections were per
formed using oligo-deoxythymicline columns (type II, Collabo
rative Research, Bedford, M A ); 10 ju-g of total R NA were
blotted on Hybond N + (Amersham, Aylesbury, U K ) according
to the procedure recommended by the manufacturer, after
glyo.xylat.ion (McMaster and Carmichael, 1977) and size frac
tionation on 1% agarose gels. To reconfirm equal loading, the
blots were subsequently hybridized to ribosomal RNA probes.
Construction ofcDNA and subtraction libraries
cDNA libraries were constructed from 5 ¡ag of oligodeoxythy
midine-selected RNA using a cD N A cloning kit (Invitrogen,
San Diego, CA). For production of the subtracted library a
sub tractor kit was used (Invitrogen). Construction was per
formed as described before (Weterman at aI., 19936).
Hybridization
D N A probes were radiolabeled using the multi prime label
ing method as recommended by Amersham. Hybridizations of
cDNA libraries were performed as described ( Weterman et ciL,
19936). Hybridizations of Northern blots were performed
according to the method of Church and Gilbert (1984) with the
addition of 0.1 mg denatured herring sperm D N A /m l of
hybridization mixture. Hybridization of Southern blots was
carried out as recommended by the manufacturer (Amer
sham). Conditions of lower stringency were obtained by
lowering the temperature for hybridization and washes down
to 15°C below the optimal temperature.
(C (AGAT )GCCAGAAGAACACTGTTGCTCTTGGTGG.ACGGGCCCAGAGGAA
03
J.12
TTCAGAGTTAAACCTTGAGTGCCTGCGTCCGTGAGAATTCAGC ATG GAA TGT CTC TAG TAT TTC
Glu Cya Leu Tyr Tyr Pha
139
166
CTG GGÀ TTT CTG CTC CTG GOT GCA AGA TTG CCA CTT GAT GCC GCC AAA CGA TTT
Leu Gl y Phe Leu Leu Leu Ala Ala Arg Leu Pro Leu Asp Ala Ala Lya Arg Phe
,193
220
CAT GAT GTG CTG GGC AAT GAA AGA CCT TCT GCT TAC ATG AGG GAG CAC A AT CAA
His Asp Val Leu Gly Ann Glu Arg Pro Ser Ala Tyr MET Arg Glu Mia Aon Gin
247
274
TTA AAT GGC TGG TCT TCT GAT GAA A AT GAC TGG A AT GAA AAA CTC TAC CCA GTG
Leu Aan Glv Trp Ser Ser Asp Glu .Ran Aap Trp Aan Glu Lys Leu Tyr Pro Val
301
32 7
TGG AAG CGG GGA G AC ATG AGG TGG AAA A AC TCC TGG AAG GGA GGC CGT GTG CAG
Trp Lys Arg Gly A0p MET Arg Trp Lya Aari Ser Trp Lyra Gly Gly Arg Val Gin
355
382
GCG GTC CTG ACC AGT GAC TCA CCA GCC CTC GTG GGC TCA AAT AT A ACA TTT GCG
Ala Val Leu Thr Ser Asp Ser Pro Ala Leu Val Gly Ser Atan H e Thr Phe Ala
409
436
GTG AAC CTG ATA TTC CCT AGA TGC CAA AAG GAA GAT GCC AAT GGC AAC ATA GTC
Val Asn Leu lie Phe Pro Arg Cyg Gin Lye Glu Asp Ala Aon Gly Awn lie Val
463
490
TAT GAG AAG AAC TGC AGA AAT GAG GCT GOT TTA TCT GCT GAT CCA TAT CTT TAC
Tyr Glu Lys Aan Cys Arg A a ri Glu Ala Gly Leu Ser* Ala Asp Pro Tyr Val Tyr
517
544
AAC TGG ACA GCA TGG TCA GAG GAC AGT GAG GGG GAA AAT GGC ACC GGC CAA AGC
Aan Trp Thr Ala Trp Ser Glu Asp ser Aap Gly Glu Awn Gly Thr Gly Gin Ser
571
S9B
CAT CAT AAC GTC TTC CCT GAT GGG AAA CCT TTT CCT CAC CAC CCC GGA TGG AGA
Hia His Aeri Val Phe Pro Asp Gly Lys Pro Phe Pro Hi a Hiss pro Gly Trp Axq
625
652
AGA TGG AAT TTC ftTC TAC GTC TTC CAO ACA CTT GOT CAG TAT TTC CAG AAA TTG
Arg Trp Asn phe lie Tyr Val Phe H:Uj Thr Leu Gly Gin Tyr Phe Gin Lya Leu
679
706
GGA CGA TGT TCA QTG AGA GTT TCT GTG AAC ACA GCC AAT GTG ACA CTT GGG CCT
Gly Arg Cya Ser Val Arg Val Se r Val Aan Thr AI a Aan Val Thr Lem Gly Pro
7 33
7 60
CAA CTC ATG GAA GTG ACT GTC TAC AGA AGA CAT GGA CGG GCA TAT GTT CCC ATC
Gin Leu MET Glu Val The Val Tyr Arg Arq HLei Gly Arg Ala Tyr Val Pro 11«
787
014
GCA CAA GTG AAA GAT GTG TAC GTG OTA ACA GAT CAG ATT CCT GTG TTT GTG ACT
A} a Gin Val Lya Asp Val Tyr Val Val Thr Aap Gin II® Pro Val Pha Val Thr
841
'
667
ATG TTC CAG AAG AAC GAT CGA AAT TCA TCC GAC GAA ACC TTC CTC AAA OAT CTC
MET Phe Gin Lye Aan Asp Arg Aan Ser Ser Arp Glu Thr Pho Lou Lym A a p Leu
m'a
922
CCC ATT ATG TTT GAT GTC CTG ATT CAT
Pro Ile KBIT Phe ftap Val Leu II© His
949
ACC ATT AAC TAC AAG TGG AGC TTC GGG
Thr lie Aan Tyr Lyei Trp Ser Phe Gly
1003
AAT CAT ACT GTG AAT CAC AGO TAT GTG
Aan Hio Thr Vai Atan ili a Thr Tyr Val
1057
ACT GTG AAA GCT GCA GCA CCA GGA CCT
Thr Val. Lya Ala Ala Ala Pro Gly Pro
' 1111
CCT TCA AAA CCC ACC CCT TCT TTA GGA
Pro Ser Lye Pro Thr Pro Ser Leu Gly
1.16 5
AGT AGG ATT CCT GAT GAA AAC TGC CAG
Ser Arg ile Pro A«p Glu Afein Cya Gin
1219
ACC ATC ACA ATT GTA GAG GGA ATC TTA
Thr H e Thr ile Val Glu Gly H e Leu
GAT.’ CCT1 AGC CAC TTC CTC AAT TAT TCT
Awp Pro Ser Hi,« Phe Leu A « n Tyr 3«sr
976
GAT AAT ACT GGC CTG TTT GTT TCC ACC
Aap Aan Thr Gly Lem ph<3 Vai Ser Thr
1030
CTC AAT GGA ACC TTC AGC CTT AAC CTC
Leu Asm Gly Thr Phe Sesr Leu A a n Leu
1084
TGT CCG CCA CCG CCA CCA GCA CCC AGA
Gya Pro Pro Pro Pro Pro Pro Pro Arg
1137
CCT GCT GGT GAC AAC CCC CTG GAG CTG
Pro Ala Gly .Aap Aan Pro Lau Glu Leu
1192
ATT AAC AGA TAT GGC CAC TTT CAA GCC
Ile Afin Arg Tyr Gly Hiei Phe Gin Aia
’
1246
GAG GTT AAC ATC ATC CAG ATG ACA GAC
Glu Val An n Ile île Gin MET Thr A tip
127 3
CCT GAA
Pro Glu
1327
GAG GTC
Glu Val
1301
TGC AGC
Cysj Ser
1300
GTG ACC
Val Thr
.1354
ACC TGC
Thr Cyei
.1407
TGT CTG
Cya Lfâu
GTC CTG ATG CCG GTG CCA TGG
Val Leu MET Pro Val Pro Trp
TGC CAA GGG AGC ATT CCC ACG
Cya Gin Gly Ser Ila Pro Thr
GAG ATC ACC CAG AAC ACA GTC
Glu Ila Thr Gin Afin Thr Val
AGC TCC CTA ATA GAC TTT GTC
Ser Sor Leu Ile A cip P ha Val
TGT ACC ATC ATT TCT GAC CCC
Cya Thr 1.1© Ile Ser Aap Pro
CCT GTG GAT GTG CAT GAG ATG
Pro Val A «p Val Aililp Glu MfiT
143 5
1462
CTG ACT GTG AGA CGA ACC TTC AAT GGG TCT GGG ACG TAC TGT GTC» AAC CTC ACC
Leu Thr Val Arg Arg Thr phe Aan Gly Ser Gly Thr Tyr Cya Val Aian Leu Thr
1409
1516
CTG GGG G AT GAC ACA AGC CTG GCT CTC ACG AGC ACC CT0 ATT TCT GTT CCT GAC
Leu Gly Aep A tip Thr Sor Leu Ala Lau Thr ilcit: Thr Leu Ilei S *;r Val Pro Aïip
1543
1570
AGA GAC CCA GCC TCG CCT TTA AGG ATG GCA AAC AGT GCC CTG ATC TCC GTT GGC
Arg Aap Pro Aia See pro Leu Arg HET Al?* Aan £ler Ala Leu H e Ser Val Gly
1597
.1624
TGC TTG GCC ATA TTT GTC ACT GTG ATC TCC CTC TTG GTG TAC AAA AAA CAC AAG
Cya Leu Ala Ile Phe Val Thr Val Ile Sor Leu Leu Val Tyr Ly a Lya fii u Lya
1651
1678
GAA TAC AAC CCA ATA GAA AAT AGT CCT GGG AAT GTG GTC AGA AGC AAA GGC CTG
Glu Tyr Aan Pro Ile Glu Aon Ser Pro Gly Aan Vai Vai Arg Ser Lys? Gly Leu
1705
'
1732
AGT GTC TTT CTC AAC CCT GCA AAA GCC GTG TTC TTC CCG GGA AAC CAG GAA AAG
Ser Val Phe Leu Aen Arg Ala Lya Aia Val Phe Phe Pro Gly Asm Gin Glu Lya
1759
1790
GAT CCG CTA CTC AAA AAC CAA GAA TTT AAA GGA GTT TCT T AA.ATTT CG A C C TT CiTTT C
Aap Pro Leu Leu Lys Aan Gin Giù Phe Lyu Gly Val Bear .
1825
1861
TG.
A AG C TC A CT Tï TC A G TG C CA T TGA T G TG AG AT G T G CT G G AG TG G CTA TTA A C CT T T T T TT C C T AAA GAT
1$96
1932
TATTGTTAAATAGATATTGTGGTTTGGGGAAGTTGAATTTÏTTATAGCTIAAATGTCATTTTAGAGATGGG
1967
2003
GAG AGGGAT T AT ACTGC AGGC AG CTT C AG CC AT GTTG TG AAACTG A ÏAAA AG CA ACTT AG C AAGG CTT CTT
2030
2074
ttcattattttttatgtttcacttataaagtcttaggtaactagtaggataoaaacactgtgtcccgagag
2109
2145
T AAGG AG AG AAG CTACT ATTG ATT AG AG CCT A.ACCC AGGTT A ACTGC AAG AAG ACGCCGGAT ACTTTC AGC
2180
2216
tttccatgtaactgtatgcataaagccaatgtagtccagtttctaagatcatgttccaagctaactgaatc
2251
2287
CC ACTTC AAT AC A CACTC ATG AA CT C CTG A TG G AA C AA TAACAG GCCC AAG C CTG TGG T ATG ATG TG C A CA
2 322
2 350
cttgctagactcagaaaaaatactactctcataaatgggtgggagtattttggtoacaacctactttgctt
FiG U.RE 2 - Comp le te n ucl eotid.e sequenee of nmb (accession
number X76534 EMBL human nmb RNA) and translated pre
dicted protein. Brackets indicate the 5' end of the various PCR
reactions,
2393
2429
ggctgagtgaaggaatgatattcatatattcatttattccatggacatttagttagtgctttttatatacc
2464
2 500
aggcatgatgctgagtgacactcttgtgtatatttccaaatttttgtatagtcgctgcacatatttgaaat
2535
2571
CATATATTAÀGACTTTCCAAAG ATG AGGTCCCTGGTTTTTCATGGCAACTTG ATC AGT AAGG ATTTCACCT
2606
2642
ctgtttgtaactaaaaccatctactatatgttagacatgacattctttttctctccttcctgaaaaataaa
GTGTGGGAAGAGAC {A )n
75
EXPRESSION OF A N OV EL G E N E IN HUM AN M E L A N O M A
DNA sequencing and computer analysis
A set of deletion clones was constructed from cDNA inserts
longer than 400-500 bp using the erase-a-base system (Promega, Madison, WI). DNA fragments were ligated into
M13mpl8, M13mpl9, pTZ or pGEM vectors and sequenced
according to the dideoxy method as described by Sanger et a l
(1980). Sequences were determined from both strands of the
cloned cDNAs. The sequences of PCR products were deter
mined using 3 independent PCR reactions. Searches for
known sequences were performed using the Genbank and
EMBL databases (Devereuxe/ al., 1984; Pearson and Lipman,
1988), Searches for motifs, alignm ents and structure predic
tions were perform ed using the C A M M S A programs M O
T IFS, P IL E U P , C L U S T A L V , B E S T F IT , P E P T ID E S T R U C T U R E , P L O T S T R U C T U R E and M E M B R A N E P R O P E N
SIT Y , which are all part o f the W isconsin Package V 7.0
(Devereuxef/a/., 1984).
Primer extension
A 149-bp A v a ll- N d e l restriction fragm ent (located at posi
tion 331-480) was used as a prim er. The extension reaction
using reverse transcriptase was based on standard protocols
(Sam brook et a l 1989); 250 ng o f double-stranded c D N A was
end-labeled using -y^P-ATP (A m ersham ) (specific activity
3 x 107 cpm/jxg), then 4 x 105 cpm were added to 10 jxg of
oiigo(-deoxythymidine)-selected M V 1 R N A . A fte r denaturation for 10 m in at 85°C, annealing was perform ed at 45°C
overnight. The extension reaction was perform ed at 37°C for 2
hr using M oloney reverse trancriptase (B R L , G ran d Island,
N Y ), purified and analyzed on a sequencing gel containing
sequence reactions as a size m arker for the length of the
extension products.
,
3.5
b7
YFLG FLLLAARLPLDAAK..RFHDVLGNERP SAYMREH NQLNGWS SDEN D 52
ft
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CLLHLAVIGALLAVGATKVPRNQDWLGVSR..... ,*.QLRT.... KA 42
53 WNEKLYPVWKRGDMRWKNSWKGGRVQAVLTSDSPALVGSNITFAVNLIFP 102
II ’ III
• : ‘ : • : 51I : I I • I
: ** I • I • I : I • I ■I * • • I II
43 WNRQLYP. .EWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFP 90
103
RCQKEDANGNIVYEKNCRNEAGLSADPYVYNWTAWSEDSDGENGTGQSHH
ft
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p
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152
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91 GSQKVLPDGQVIWVNN........ TriNGSQVWGGQPVYPQETDDAC. 12 9
153 NVFPDGKPFPHHPGWRRWNFIYVFHTLGQYFQKLGRCSVRVSVNTANVTL 202
•
ft
ft
•
•
*
ft
ft
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I ft
ft
ft
ft f
I
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*
130 .IFPDGGPCPSGSW5QKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAML 178
203 GPQLMEVTVYRRHG.RAYVPIAQVKDVYVVTDQIPVFVTMFQKNDRNSSD 251
: ;
179 GTHTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPF5VSVSQLRALDGGN 228
*
♦
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*
9
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4
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252 ETFLKDLPIMFDVLIHDPSHFLNYSTINYKWSFGDNTGLFVSTNHTVNHT 301
::
229 KHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAPVVTHT 278
ft
ft
ft
ft
•
ft
ft
ft
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«
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ft
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ft
. .
302 YVLNGTFSLNLTVKAAAP...................!.........
ft
ft
ft
V
ft
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ft
»
319
ft
279 YLEPGPVTAQWLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPT 328
•
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ft
•
m
320 ___GPCPPPPPPPRPSKPTP........................... 336
•
ft
ft
ft
ft
ft
•
I
ft
329 TEVVGTTPGQA PTAEPSGTT SVQVPTT EVISTAPVQMPT AE STGMTPEKV 37B
ft
ft
ft
ft
m
337 ..................... SLGPAGDNPLELSRIPDE. ..NCQINR 360
ft
ft
■
*
f
Cloning o f the 5' end o f nm b cDNA
The 5' end o f nmb was cloned using the am plifinder kit
(Clontech, Palo A lto , C A ). Basically, after oligo-deoxy thym i
dine-primed first-strand synthesis using 2 jxg of poly-A+selected M V1 R N A as a tem plate, an anchor adaptor prim er
was ligated to this single-stranded c D N A and am plified. For
amplification, this prim er and a specific prim er (located at
457-481) were used as P C R primers (5 cycles, using 10 pm ol of
each prim er and Taq polymerase). For further P C R am plifica
tion, another specific prim er was used (located at position
403-427), 35 Cycles o f am plification were performed: denaturation for 45 sec at 94°C, annealing for 45 sec at 60°C, and
extension for 90 sec at 72°C. F inal extension was allowed to
proceed for 7 m in. P C R products were analyzed on an agarose
gel, treated with T4 D N A polymerase to create blunt ends and
cloned into a blunt-end vector.
ft
4
429 ELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYR 478
361 YGHFQATITIVEGILEVNIIQMTDVLMPVPWPESSlilDFWT 3GSIPTE 410
i
ft
0
ft
ft
ft ft
ft ft
ft •
ft ft
ft
I
I
*
+f
•
*
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ft
479 YGSFSVTLDIVQG.....IESAEILQAVPSGEGDAFELTVS
Q3GGLPKE 522
Transfection
Transfection was perform ed in the B L M cell line using
lipofectin (B R L ) and 20 jxg of p Z IP n e o (C epko et a l 1984;
D o tto et a l, 1985) carrying the G418 resistance gene and a
c D N A clone (416-2656) or the resistance gene alone. Stable
transfectants were selected in the presence o f L m g/m I of G418
in the culture m edium . A fter selection, cells were grown in the
presence of 0,25-0.5 m g /m I G418.
,
411 VlClTIISDPT EITQNTV SPVDVDEf&LTVRRTF NGSGTVlCVNLTLGD 459
.
i
ft ft ft
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523 ACflEISSPGjC 2PPAQRl®PVLPSPAla3LVLHQlLKGGSGTYldLNVSLAD 572
460 DTSLALTSTLISVPDRD. ..PASPLRMANSALISVGCLAIFVTVISLLVY 506
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573 TNSLAWSTQLIMPVPGILLTGQEAGLGQVPLIVGILLVLMAWLASLIY 622
507 KKHKEYNPIENSPGNWRSKGLSVFLNRAKAVFFPGNQEKDPLLKNQEF 555
•
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623 RRR..LMKQDFSVPQLPHSS..SHWLRLPRIFCSCPIGENSPLLSGQQV 667
Figuris 3 - Alignment of pM el l 7 (h) to the predicted protein of
nmb (pnmb; a). Conserved cysteines are shown in boxes.
100
i ■i ••
5.0
In vivo assay for metastascs
Approximately 3 x 106 cells were inoculated sx. into nude
mice. Tum or volum es were m easured weekly and mice were
m aintained for 3 m onths unless the tum or size interfered with
their health. A fte r autopsy, the lungs were formalin-fixed, and
200
300
-100
500
i i i i I i i i i i I i i i I i i i i i i i i i I i i r i r i i i i I t i i ■i ' i i t I i i i i i
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------
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" 11
c-p— c— C-
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->-1______ I_____ I__ LJUUU_________________ U
i i i i i i i i i I t t i i i i t i i j i i i i i i i i i I i i i i i i i i t |i i i i t i i i t |i i i i i
100
200
3QQ
40U
5D0
sequences In order to make it a biologically
sequence is marked with an asterisk.
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79
EXPRESSION OF A N O V E L G E N E IN H U M A N M E L A N O M A
A
B
VOLUME (om3)
VOLUME (ofii3)
WEEKS
BLM Hpo
BLM M o 1
BLM *141
BLM 2 1 »
F ig u r e
BLM 21.6
BLM Hpo
BLM n to 1
BLM n«o 7
BLM 91.6
BLM 21B
BLM S1.1t
BLM n«0 10
10 - Growth curves of the tumors from parental, neomycin- and nwb-transfected cell lines in nude mice. Values are given as a
mean of all animals tested, (a) and (b) represent separately performed experiments. The values were based on 5 animals per cell line
except for 21.19 (4 animals) in (a) and 10 (21.8, neol, neol6), 9 (21.19, BLM, neo7) or 7 animals in the case of 21.5 in (b).
'FABLE I - RATES O F SPONTANEOUS LUNG METASTASIS AFTER S.G
INOCULATION OF NEOMYCÎN-, AW/i-TRANSFECTANTS, O R PARENTAL
CONTROLS IN N UDE MICE
Cell line
A
(%)
B
(%)
Parental BLM
neo 1
neo 7
neo 16
21.5
4/5
2/5
N.D.
N.D.
1/5
1/5
1/4
80
40
5/9
5/9
8/10
5/10
2/7
6/10
'55
'55
80
50
’29
60
75
21.8
21.19
6/8
The parental cell line was mock-transfected with lipofectin
without adding D N A . A and B represent separately performed
experiments.
*
parental control ccll lines were readily observed. In particular,
clone 21.5 had a tendency to cluster at lower densities (Fig. 9).
Growth rales in vitro were comparable. Growth rates in vivo
and metastatic potential were examined in 2 separate sets of
experiments. U pon inoculation in nude mice, tum or growth
was delayed in the case of d o n e 21.5, and to a lesser extent of
clone 21.19 as compared to control cell lines (Fig. 10). After
autopsy of the animals, the lungs were microscopically exam
ined for métastases. In both sets of experiments, metastatic
potential of 21.5 transfectants was reduced when compared to
either parental or neomycin-resistent control transfcctant cell
lines (Table I) while for the other 2 nm/>transfectants variable
results were obtained.
D IS C U S S IO N
U pon comparison of expression in highly and lowly meta
static cell lines, several cDN As were isolated showing a
differential expression in the lowly and highly mctastatic
human m elanom a ccll lines and derived xenograft lesions.
c D N A clones that showed a difference in expression of only 2to 4-fold appeared to be related to growth and metabolism, e.g.
ribosomal protein L8. Ribosomal cD N As were also isolated
from a tumor-enriched colon subtraction library, in which
expression was detected in both well- and poorly-diifcrcntiated cells. Increased expression correlated with progression in
2 pairs of cell lines derived from primary and metastatic lesions
of the same patient (Kondoh et a l, 1992), Although the aim of
this subtraction library was to enrich for sequences strongly
expressed in the highly metastatic cell line, in 2 of the isolated
cD N A s the pattern seen was the opposite of what was
expected. Since an excess of MV1 sequences was used to
hybridize to M V 3 sequences and the labeling o f M V 1 se
quences by photobiotinylation, hybridization and separation of
hybridized and non-hybridized c D N A s was not com plete, part
of this library consists of cD N A s strongly expressed in the
low-metastatic M V 1 cell line.
Sequence analysis o f nmb, which showed expression only in
the low-metastatic hum an m elanom a ccll lines and xenografts,
revealed that this clone represents a novel gene. Based on the
presence o f a signal sequence, a hydrophobic area o f 27
amino-acid residues, bordered by charged residues, which
might cross the m em brane in a single helical span, the
prediction of integral m em brane proteins by membrane propen
sity programs for both signal sequence and transm em brane
domain, and the homology with the precursor of p M e ll7
melanocytic-specific protein (Kw on et a l, 1991) which also
shows a transm cm brane dom ain in this region, we assume that
this d o n e is encoding a transm em branc glycoprotein. Since
M e l 17 was described as a m em ber o f a family to which gp75
and tyrosinase also belong, which are both prom inently ex
pressed in melanocytic lesions, we also aligned our clone to
these sequences and 5 regions of homology were detected (Fig.
11). Overall identity and hom ology was 17.6% and 41.7% in
the case o f gp75, and 16.7% and 40.8% in the case of
tyrosinase. A ll are characterized by a short non-coding region
at the 5' end o f the messenger, a signal peptide, and a
transmcmbrane dom ain near the 3' end. Nearly all cysteines
are conserved between tyrosinase and gp75 (C h in tam an e n i et
a l, 1991), whereas all cysteines in the region preceding the
transmcmbrane region are conserved between p M e ll7 and
pnm b (Fig. 3). A lth o u g h these 4 proteins clearly belong to one
family, gp75 and tyrosinase are m ore sim ilar to each other than
to p M e ll7 or pnm b, and vice versa. Tyrosinase is the key
enzyme in the synthesis of m elanin pigm ent. A lthough tyrosi
nase, gp75 and p M e ll7 are described as melanocyte-speciiic,
and tyrosinase and gp75 are localized to the melanosomes,
nmb is expressed in other tissues and tum or cell lines as well.
Therefore, it cannot be present exclusively in the melanosomes.
Screening of several other ccll lines, rat organs and tumors
indicated that the nmb gene has a restricted expression
pattern, is not specific for the melanocytic lineage, and,
secondly, is not well conserved d u rin g evolution o f vertebrates.
A lthough nmb R N A expression is not significantly higher in
pooled specimens of nevocellular ncvi than in m elanom a
metastases, no final conclusions can be drawn about its
potential as a protein m arker for particular stages in m elano
cytic progression, since R N A levels do not necessarily predict
WETERMAN ETAL.
80
C
C
0
H
V
A
D
D
S
T
W
V
S
V
L
L
K
E
G
G
N
A
V
N
L
L
S
E
W
W
N
D
H
H
Q
L
R
R
P
P
L
Y
L
I
F
H
T
M
L
L
F
F
L
L
A
D
R WE Q E
R L E K D
L H D
L
V L I H D
I
K
P
P
D s
F s
L I
ILS
F
V
M
V
(1)
Tyr
gp75
Pm ell7
Pnmb
(189)
<196)
(209)
(234)
Tyr
(436)
gp75
Pm ell7
Pnmb
(1)
(2)
(2 )
(147)
(153)
(158)
(184)
H F P R A
3 F P R Q
K V P R N
- R F
K
Tyr
gp75
Pm ell7
Pnmb
F
F
F
F
L
(426) L
(558) L
{437) L
L
L
L
L
P
T
R
K
G yId Y
G y T y
A D T N
G D D T
1 y l q d
E I 2 WP
S L A V V
s L A L T
S
S
s
s
M
R
R
R
D
R
T
T
P
E
Q
L
Q
Q
S
S
K
E
G
H
P
Ü F N D I N I y D L F V WMH y
L
M
Y
F
T
P£ F E N I S I y N y F V W T H y
T H T ME V T V Y H R R G S R 9 y
P Q L ME V T V y R R H G - R H y
GD E N F T 1
L Q E P S F S h
L A E A D h s y
S T I N y
L N
|e K Q
(486) A N Q
(635) rs] N S
(524) E K D
p y w D W
p Y w N F
w D F
T
w S F
K
-
m*
P
P
P
P
L
L
L
L
L
L
L
L
M
S
K
R D
A T
G D
G D
E K E D y
~ T D Q y
G 0 M
E, - -
- Multiple alignment of tyrosinase, gp75, p M e l!7 and pnmb. Positions were based on mature peptides. The regions shown
were part of the alignments of the complete sequences, as performed by the PILE UP or CLUSTALV alignment methods.
F i g u r e 11
protein data. Earlier studies showed that, although an elevated
R N A expression of calcyclin was detected in melanoma
metastases, this did not lead to a m uch higher expression of the
protein since often only a small percentage o f the tum or cells
within such a lesion was stained (W eterm an et a l, 1992,
1993a).
Since the most im portant step in tum or progression is
acquisition of the metastatic phenotype, we were interested to
determine whether nmb could reduce the metastatic potential
of a highly metastatic «mfr-negative m elanom a cell line. Based
on their expression pattern of nmb , calcyclin and thymosin
(3-10, 3 /wiZ?~transfectants (21.5, 21.8 and 21.19) were chosen
for further characterization. Calcyclin levels varied in the
transfectants, which was not surprising since other experi
ments using various m elanom a cell lines showed that intrinsic
calcyclin m R N A levels varied widely after transfection experi
ments or changes within the cell lines themselves (unpublished
observations). In a prelim inary series of experiments, a ten
dency toward growth delay and reduction o f metastatic poten
tial was clearly present in the case of transfectant 21.5. The
other transfectants tested, 21.8 and 21.19, showed varying rates
of metastasis, possibly due to lower protein levels, independent
of the R N A level, which might condition the metastatic
potential. It should be noted that a partial c D N A was
transfected which lacked the signal peptide. Nevertheless,
since the cytoplasmic part o f the peptide was present in the
transfectants, these observations are probably meaningful and
will be extended in further studies.
ACKNOWLEDGEMENTS
The use of the services and facilities of the Dutch National
Expertise Center C A O S /C A M M , under grants S O N 326-052,
and STW NCH99.1751 is gratefully acknowledged. W e thank
Mr. G. Stoopen, Mr. J. Berkeljon, Ms. I. Cornclisscn, Mr. K.
Jansen and Ms. J. Lemmcrs for their technical assistance. This
work was supported by the D utch Cancer Society, grant
N U K C 89-08.
REFERENCES
A k s le n , L.A. and M o r k v e , O., Expression of p53 protein in cutaneous
R ie t h m u lle r , G., De novo expression of intercellular-adhesion mol
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