Download Molecular Cloning and Characterization of an

[CANCER RESEARCH 48, 2955-2962, June 1, 1988)
Molecular Cloning and Characterization of an Antigen Associated with Early
Stages of Melanoma Tumor Progression1
Hak Hotta, Alonzo H. Ross,2 Kay Huebner, Masaharu Isobe, Sebastian Wendeborn, Moses V. Chao,
Robert P. Ricciardi, Yoshihide Tsujimoto, Carlo M. Croce, and Hilary Koprowski
The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 [H. H., A. H. R., K. H., M. I., S. W., R. P. R., Y. T., C. M. C., H. KJ; Department of
Microbiology, Kobe University School of Medicine, Kobe 650, Japan [H. H.]; and Department of Cell Biology and Anatomy, Cornell University Medical College, New
York, New York 10021 [M. V. CJ
ABSTRACT
The melanoma-associated
antigen ME491 is expressed strongly during
the early stages of tumor progression. The ME491 gene was molecularly
cloned by means of DNA-mediated gene transfer followed by screening
a X genomic library with human repetitive Alu sequences as a probe. The
cloned DNA, after transfection into mouse I .-cells, generated a protein
with characteristics that were indistinguishable in Western blot analysis
from the ME491 antigen expressed by human melanoma cells. Repeatfree subfragments of the cloned DNA were used for further studies. By
Northern blot analysis, the subfragments detected a single 1.2-kilobase
mRNA in the transformants and various human melanoma cell lines.
ME491 complementary DNA clones were then obtained by probing a
melanoma complementary DNA library with the genomic subfragments.
Nucleotide sequence analysis of the cloned complementary DNA indi
cated that the ME491 antigen consists of 237 amino acids (M, 25,475)
with four transmembrane regions and three putative JV-glycosylation
sites. No significant structural homology was observed with other proteins
thus far reported. We observed that the amounts of mRNA varied greatly
with different melanoma cell lines. Southern blot analysis revealed no
amplification or rearrangement of the ME491 gene in the human mela
noma cell lines tested, including both high and low expressors of this
antigen. The ME491 gene has been mapped to chromosome region
12pl2—»12ql3 by somatic cell hybrid analysis and more narrowly local
ized to 12ql2-»12ql4 by in situ hybridization.
INTRODUCTION
Because melanoma is a pigmented, cutaneous lesion, it has
been possible to observe the development of the disease and to
develop a detailed model of melanocytic tumor progression (1).
A large number of MAbs3 against human melanomas have been
prepared to analyze the stages of progression (2—4).Most of
these MAbs bind to both melanoma and the benign melanocytic
lesion, the nevus, but not to normal tissue melanocytes. There
are also a few MAbs that bind only to melanoma but not to
dysplastic nevi (3). Although the tissue distributions and mo
lecular weights have been determined for many of these MAAs,
the biological functions and relevance to tumor progression are
little understood. Among the human MAAs, only two antigens,
M, 97,000 protein (4) and NGF receptor (5, 6), have been
characterized thus far by means of gene cloning.
ME491 antigen is a MAA which is a marker for the early
stages of tumor progression of human melanoma (7, 8). It is
not detected on normal tissue melanocytes but is strongly
expressed in dysplastic nevi and radial growth phase primary
Received 2/24/88; accepted 3/25/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by NIH Grants NS-21716, CA-25874, CA-10815, and RR-05540
and grants from the National Neurofibromatosis Foundation and the Dysautcnomia Foundation.
2To whom requests for reprints should be addressed, at The Worcester
Foundation for Experimental Biology, 222 Maple Ave., Shrewsbury, MA 01S4S.
3 The abbreviations used are: MAb, monoclonal antibody; MAA, melanomaassociated antigen; SDS, sodium dodecyl sulfate; cDNA, complementary DNA;
SSC, standard saline-citrate.
melanomas. The antigen expression, however, is weaker or
sometimes even negative in more advanced stages of melanoma
such as vertical growth phase primary melanoma and metastatic
melanoma. Because of its stage-specific expression, this antigen
has been considered as one of the interesting MAAs. ME491
antigen is also strongly expressed in adenocarcinomas of the
colon and prostate, and to a lesser degree in some other human
tumors (8, 9). It is detected even on undifferentiated colorectal
carcinomas which lack most other gastrointestinal tumor-as
sociated antigens (9).
Biochemical analyses have shown that ME491 antigen is a
membrane-bound glycoprotein present both inside the cell and
at the cell surface the molecular weight of which ranges from
30,000 to 60,000 with a single core protein of about 20,000
(10). This heterogeneity is apparently due to A'-linked glycosylation since tunicamycin eliminates this heterogeneity. The
NI I..-terminili amino acid sequence of ME491 antigen has been
determined for the first 20 amino acids and shows no homology
with known proteins (10). Other investigators have independ
ently obtained MAbs which detect very similar or identical
antigens and have characterized them in pathological and clin
ical studies (11-13).
As a step toward understanding the significance of ME491
antigen for tumor progression, we have molecularly cloned the
human gene and cDNA-encoding ME491 antigen. In the pres
ent study, we describe the molecular cloning, expression, and
chromosomal localization as well as nucleotide sequence analy
sis of this antigen.
MATERIALS
AND METHODS
DNA-mediated Gene Transfer to Mouse L-Cells
High molecular weight DNA was isolated from human melanoma
cell line A87S and from human cervical carcinoma cell line HeLa, both
of which express ME491 antigen.4 The DNA was cotransfected with
purified herpes simplex virus thymidine kinase gene into Uk cells by
the calcium phosphate precipitate method as described (5, 14). After
selection in a IS /¿g/ml-hypoxanthine-1 ¿tg/mlaminopterin-5-Mg/ml
thymidine medium, resulting tk* colonies were screened with an immunological rosette assay (S) in which cells are incubated with MAb to
ME491 antigen and then a second antibody (rabbit anti-mouse IgG)
coupled to erythrocytes. Positive colonies were cloned and grown in
bulk to extract high molecular weight DNA. To reduce the amount of
extraneous human DNA, a second round of transfection was performed
using DNA extracted from one of the primary transformants (MES).
Genomic DNA Library
High molecular weight DNA from one of the secondary transformants (ST2-3) was partially digested with .Vu«3A
restriction enzyme and
fractionated by 10 to 40% sucrose gradient uItracent rifugni ion (IS).
Fragments from 15-23 kilobases were ligated with both arms of EMBL
3 DNA (Promega Dioice, Madison, WI), packaged into phage particles
using a commercial packaging extract (Giga-Pack Gold; Stratagene,
' Unpublished observations.
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MOLECULAR CLONING OF ME491 ANTIGEN
San Diego, CA), and then inoculated onto Escherichia coli NM539 to
make a genomic DNA library. This library was screened for human
DNA sequences using human repetitive Alu sequences (16) as a probe.
Southern and Northern Blot Analyses
Total cellular DNA isolated from cultured cells and tissues was
digested with appropriate restriction enzymes, fractionated by agarose
gel (0.8 or 1%) electrophoresis, and transferred to nitrocellulose filter
paper (15). To identify human DNA sequences present in the trans
formed mouse cells, the transferred DNA was hybridized with nicktranslated "P-labeled human repetitive Alu sequences (16) in 50%
formamide, 5x SSC (Ix SSC is 0.15 M NaCl plus 0.015 sodium citrate),
5x Denhardt's solution, and 100 tig of salmon sperm DNA per ml at
42"C for 20 h. To identify ME491 sequences in human and mousehuman hybrid DNAs, filters were hybridized to radiolabeled pMel-17
DNA in the same hybridization mix without formamide at 65°C.The
blots were washed with 0.2x SSC-0.1% SDS at 65°Cunless otherwise
stated, dried, and exposed to Kodak XAR-5 film at -70°C.
For regional localization of the ME491 gene on chromosome 12,
three mouse-human hybrids which had previously been observed to
retain partial chromosomes 12 (22, 24) were used. The presence of a
group of chromosome 12 DNA markers, including the ME491 gene,
was determined in order to determine which parts of chromosome 12
were present or absent in the three hybrids. The chromosome 12-linked
probes for T4, K-ros-2, homeobox gene C8, HPV 18 integration site
flanking region 7.16.1, and insulin-like growth factor I have been
described (22, 24-27).
In Situ Hybridization. Metaphase spreads were prepared from nor
mal human male lymphocyte cultures stimulated with phytohemagglutinin for 72 h. The 1.1-kilobase subcloned DNA fragment (pMel-17)
was 'H labeled by nick-translation and hybridized to the metaphase
spreads as described elsewhere (26, 28, 29).
RESULTS
DNA-mediated Gene Transfer to Mouse Ltk~ Cells. After
Polyadenylated RNA isolated from cultured cells was denatured,
fractionated by electrophoresis through 1.5% agarose/formaldehyde
gel, and transferred to nitrocellulose paper (15). Filters were hybridized
with 32P-labeled probes under the same conditions used for Southern
transfection with high molecular weight DNA from human cell
lines HeLa and A875, positive colonies expressing ME491
antigen were obtained at a frequency of 1 per 10,000 tk+
blot analysis.
colonies. A stable primary transformant, MES, was derived
from HeLa DNA and was used for further experiments. In the
second round of transfection using genomic DNA from the
MES primary transformant, positive colonies were obtained at
practically the same frequency as that determined in the first
round. Southern blot analysis using human repetitive Alu se
quences as a probe revealed that secondary transformants con
tained much smaller amounts of human DNA than did primary
transformants and that two independent secondary transformants (ST1-1 and ST2-3) appeared to share some ^/«-positive
fragments (6.8- and 3.2-kilobase BamHl fragments) (Fig. 1).
The human repetitive Alu sequences did not hybridize to Ltk~
Cell Extracts and Western Blot Analysis
Cultured cells were swollen in hypotonie buffer, lysed by freezethawing, and centrifugea at 100,000 x g for 30 min as described
elsewhere (10, 17). The membrane-containing pellet fraction was ex
tracted with 0.5% Nonidet P-40-140 HIM NaCl-10 HIM NaF-10 HIM
Tris-5 IHMEDTA-100 Kallikrein lU/ml aprotinin-1 IHMphenylmethylsulfonyl fluoride, pH 7.5, for 30 min at 4°C.The sample was then
centrifuged at 100,000 x g for 30 min, and the supernatant was analyzed
by SDS-polyacrylamide gel (10%) electrophoresis. Fractionated pro
teins were transferred electrophoretically to nitrocellulose paper and
incubated with mouse anti-ME491 MAb and then 125I-labeledgoat antimouse IgG to visualize the antigen by autoradiography.
cDNA Library
Polyadenylated RNA was isolated from WM1158 melanoma cells
by oligo-deoxythymidylate chromatography (18) and used to construct
a cDNA library in Xgtll as described (19) with certain modifications.
Briefly, 25 ^g of polyadenylated RNA were used for the synthesis of
the first strand using reverse transcriptase and oligodeoxythymidylate
(chain length, 12-18 residues) as primer. Second strand synthesis was
performed with E. coli DNA polymerase I and E. coli RNase H as
described (20). The double strand cDNA was made blunt ended using
T4 DNA polymerase and methylated with EcoRl methylase. Phosphorylated KcoRl linkers were ligated to the ends of the double strand
cDNA, and excess linkers were removed by EcoRl endonuclease. The
cDNA was ligated into the £coRIsite of Xgtl 1 vector DNA, packaged
in vitro, and inoculated onto K. coli Y1088 to make a cDNA library.
DNA Sequence Analysis
Cloned cDNAs were digested with appropriate restriction endonu
clease to generate small fragments. The fragments were subcloned into
M13mp 18 and M13mp 19 vectors (Bethesda Research Laboratory, Inc.,
Gaithersburg, MD). Single strand DNA was prepared, and the nucleotide sequences were determined by the dideoxynucleotide chain termi
nation method (21). Both strands were then sequenced.
Chromosome Mapping of the ME491 Gene
DNA. These observations indicated that the gene encoding
ME491 antigen was very close to or even included in the 6.8and/or 3.2-kilobase fragments.
Molecular Cloning of Genomic DNA Sequences Encoding
ME491 Antigen. An EMBL 3 genomic library was prepared
with DNA from one of the secondary transformants (ST2-3)
and probed with human repetitive Alu sequences. Two Alupositive recombinant clones, designated XR31 and XR33, were
obtained from 500,000 recombinant clones. Restriction enzyme
map analysis revealed that XR31 and XR33 partially overlapped
and that the overlapping regions contained the ^/«-positive 6.8and 3.2-kilobase Marnili fragments detected by Southern blot
analysis of the secondary transformants (Fig. 2). Another family
of human repetitive sequences was also mapped to the overlap
ping regions. For further studies, fragments free of human
repetitive sequences (pMel-29, pMel-17, and pMe2-13) were
subcloned into pBR322.
To determine whether these genomic DNA fragments con
tained genetic information necessary for ME491 antigen
expression, XR31 and XR33 DNA as well as a cloned mouse
DNA (XR47) as a negative control were transfected into Ltk~
cells and expression of ME491 antigen was determined by the
immunological rosette assay. Both XR31 and XR33 DNA could
transform the cells to express the antigen (Table 1), suggesting
that the gene necessary for ME491 antigen expression is located
on the overlapping region. Digestion of XR31 DNA with
Ramili or KcuRl prior to transfection completely abolished
ME491 antigen expression, whereas Hindlll digestion did not
affect antigen expression (Table 1).
To determine which subfragment(s) contains exon(s) for
ME491 antigen, Northern blot analysis was done using the
subcloned fragments as a probe. pMe 1-17(1.1 -kilobase Bamlll-
Somatic Cell Hybrid Analysis. Isolation, propagation, and character
ization of parental cells and somatic cell hybrids used in this study have
been described previously (22-24). DNAs extracted from the hybrids
and the parental cells were subjected to Southern blot analysis using a
subcloned DNA fragment (pMel-17; 1.1-kilobase BamHl-EcoRI frag
ment) as probe. This probe has been shown to hybridize to ME491
mRNA.
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MOLECULAR CLONING OF ME491 ANTIGEN
Table 1 Frequency of positive colonies after transfection with the recombinant
DNA clones
Mouse Ltk~ cells (1 x IO6) were transfected with 2 ^g of recombinant DNA
clone, 100 ng of pTK, and 15 jig of Ltk~ DNA in the calcium phosphate
precipitate. After 2 weeks in the hypoxanthine-aminopterin-thymidine
selection
medium, plates were screened by the immunological rosette assay.
kb
%of
positive
Recombinant DNA clones
colonies
50(1000)°
XR31
XR31, ««milltreated
0(500)
XR31,£coRI treated
0(200)
XR31, ffmdlll treated
45(1000)
XR33
70(1000)
XR47 (mouse DNA)
0(2000)
" Numbers in parentheses, approximate number of colonies tested.
23 —
9.4 —
6.6 —
4.4 —
pMe1-17
pMe1-29 pMe2-13
CO
m
Z
2.3 —
co
co
er
«e
co
co
OC
2.0 —
28S —
I
»
18S —
*•
1
6
Fig. 1. Detection of human DNA sequences in primary and secondary trans
formants using Alu repetitive sequences as a probe. Total cellular DNA was
digested with «umili and separated on a 1% agarose gel (20 *ig/lane). After
transfer to a nitrocellulose filter, the DNA was probed with 300 base pair Alu
human repetitive sequence. (Washing conditions were 0.5x SSC-0.1% SDS at
65*C.) (Lane 1) N2C, a primary transformant which was generated by transfection
of Ltk~ cells with total human DNA; (Lane 2) mouse I ik ; (Lane 3) a primary
transformant MES the DNA of which was used to generate secondary transformants; (Lane 4) a secondary transformant ST2-3; (Lane 5) an independently
obtained secondary transformant ST1-1-2; (Lane 6) Sill,
a partially cloned
culture with 30% antigen-positive cells; the ST1-1-2 clone was derived from this
culture.
E E
III
pMe1-29
pMe1-17
pMe2-13
I
HE
HEB
1
I
1
I
1
Fig. 2. Restriction maps of ME491 genomic clones. XR3I and XR33 were
cloned from a X genomic library prepared with secondary transformant ST2-3
DNA using Alu human repetitive sequences as a probe, n, mouse cellular se
quences which were identified by Southern blotting using Ltk~ DNA as a probe;
•.Alu human sequences; A, human repetitive sequences other than the Alu family.
lì,
H, and E, restriction sites for /fumili,EcoRl, andHindlll, respectively. pMel29, pMel-17, and pMe2-13 are fragments subcloned into pBR322. kb, kilobase.
EcoRl fragment) strongly hybridized to a 1.2-kilobase polyadenylated RNA from both a XR33 transformant (L-XR33) and
human melanoma cell line A875 (Fig. 3). Note that the tran
script was much more abundant in L-XR33 than in A875 cells.
pMel-29 (l.S-kilobase BamHl-EcoRl
fragment), but not
pMe2-13, was also shown to hybridize to the 1.2-kilobase
polyadenylated RNA from the L-XR33 transformant.
123
4
5
Fig. 3. Detection of ME491 mRNA in L-XR33 and A875 human melanoma
cells. Polyadenylated RNA was separated on a 1.5% agarose/formaldehyde gel (3
Mg/lane). After transfer to a nitrocellulose filter, the RNA was probed with pMel17 (Lanes 1, 2, and J), pMel-29 (Lane 4) or pMe2-13 (Lane 5). (Lane 1) Mouse
Ltk~; (Lane 2) A875; (Lanes 3, 4, and S) LXR33.
Identity of the Antigen Expression in L-XR33 and Human
Melanoma Cells. The antigen expressed in the L-XR33 trans
formant was biochemically characterized and compared with
that expressed in human melanoma cells. Western blot analysis
of the membrane fractions from L-XR33 and A875 melanoma
cells showed practically the same pattern, i.e., a broad band
with a molecular weight ranging from 30,000 to 60,000 (Fig.
4). This broad band has been observed in all melanoma cells
thus far tested and may be the result of extensive and hetero
geneous glycosylation (8, 10). The cytosol fraction of L-XR33
cells contained almost no ME491 antigen (Fig. 4), in agreement
with previous reports with melanoma cells (7).
Nucleotide Sequences and Deduced Amino Acid Sequences of
ME491 Antigen. A Xgtll cDNA library prepared with mRNA
from melanoma cell line WM 1158 was probed with the repeatfree genomic subfragments (pMel-17 or pMel-29), and several
overlapping clones were obtained. Fig. 5 shows the nucleotide
sequences and deduced amino acid sequences. The predicted
amino acid sequence following the first ATG codon coincides
perfectly with the NH2-terminal amino acid sequence (Nos. 1
to 20) of purified ME491 antigen except for No. 8 (Cys) which
was not previously assigned (10). The ME491 antigen consists
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MOLECULAR CLONING OF ME491 ANTIGEN
<0
o
0)
C
<o
*_
o
m
O
-Q
E
in
r-
(D
M.W.
(kDa)
200 —
3
0)
O
926843 —
25-
18 —
14 —
2
3
5
6
Fig. 4. Detection of ME491 antigen in L-XR33 and human melanoma cell
lines by Western blot analysis. Cells were separated to a membrane fraction and
a cytosol fraction. The membrane fractions, unless otherwise stated, were sub
jected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellu
lose niter. The filter was incubated with ME491 MAh and then with '"I-rabbit
anti-mouse IgG to visualize the antigen by autoradiography. (Lane 1) L-XR33;
(Lane 2) cytosol fraction of L-XR33; (Lane 3) Ltk~; (Lane 4) A875 melanoma;
(Lane 5) WM9 melanoma; (Lane 6) human neurofibroma. kDa, molecular weight
in thousands.
GCCTTGGGAAGCCCAGGCCCGGCAGCC
ATG GCG GTG GAA GGA GGA ATG AAA TGT
HetAlaValGluGlyGìy
MetlysCys
GTG AAG TTC TTG CTC TAC GTC CTC CTG CTG GCC TTT TGC GCC TGT GCA GTG GGA CTG ATT
ValLysPheLeuLeuTyrValLeuLeuLeuAlaPheCysAlaCysAlaValGlyLeuIle
GCC GTG GGT GTC GGG GCA CAG CTT GTC CTG AGT CAG ACC ATA ATC CAG GGG GCT ACC CCT
27
8
87
28
147
AlaValGlyValGlyAlaGinLeuValLeuSerGinThr IleIleGinGlyAlaThrfra
48
GGCTCTCTGTTGCCAGTGGTCATCATCGCAGTGGGTGTCTTCCTCTTCCTGGTGGCTTTT
GlySerLeuLeuProValVal IleIleAlaValGlyValPheLeuPheLeuValAlaPhe
207
68
GTGGGCTGCTGCGGGGCCTGCAAGGAGAACTATTGTCTTATGATCACGTTTGCCATCTTT
ValGlyCysCysGlyAlaCysLysGluAsnTyrCysLeuMet IleThrPheAla IlePhe
267
88
CTG TCT CTT ATC ATG TTG GTG GAG GTG GCC GCA GCC ATT GCT GGC TAT GTG TTT AGA GAT
327
LeuSerLeu IleMetLeuValGluValAlaAlaAla IleAlaGlyTyrValPheArgAsp
108
AAG GTG ATG TCA GAG TTT AAT AAC AAC TTC CGG CAG CAG ATG GAG AAT TAC CCG AAA AAC
Lys Val Met Ser Glu Phe Asn Asn Asn Phe Arg Gin Gin Met Glu Asn Tyr Pro Lys Asn
387
128
AAC CAC ACT GCT TCG ATC CTG GAC AGG ATG CAG GCA GAT TTT AAG TGC TGT GGG GCT GCT
447
AsnH1sThrAlaSerIleLeuAspArgMetGinAlaAspPheLysCysCysGlyAlaAla
148
AAC TAC ACÕ GAT TGG GAG AAA ATC CCT TCC ATG TCG AAG AAC CGA GTC CCC GAC TCC TGC
Asn Tyr Thr Asp Trp Glu Lys Ile Pro Ser Met Ser Lys Asn Arg Val Pro Asp Ser Cys
507
168
TGCATTAATGTTACTGTGGGCTGTGGGATTAATTTCAACGAGAAGGCGATCCATAAGGAG
CysHe AsnValThrValGlyCysGlyIleAsnPheAsnGluLysAla IleH)sLysGlu
567
188
GGCTGTGTGGAGAAGATTGGGGGCTGGCTGAGGAAAAATGTGCTGGTGGTAGCTGCAGCA
GlyCysValGluLysIleGlyGlyTrpLeuArgLysAsnValLeuValValAlaAlaAla
627
208
GCC CTT GGA ATT GCT TTT GTC GAG GTT TTG GGA ATT GTC TTT GCC TGC TGC CTC GTG AAG
687
AlaLeuGlyIleAlaPheValGluValLeuGlyIleValPheAlaCysCysLeuValLys
228
AGT ATC AGA AGT GGC TAC GAG GTG ATG TAG GGGTCTGGTCTCCTCAGCCTCCTCATCTGGGGGAGTGGA
747
SerIleArgSerGlyTyrGluValMetEND
237
ATAGTATCCTCCAGGTTTTTCAATTAAACGGATTATTTTTTCA
790
Fig. S. Complete nucleotide sequence of ME491 antigen and its predicted
primary amino acid sequence. MI .-terminal amino acid sequence of purified
ME491 antigen (32) is underlined. Putative recognition sites for asparaginelinked JV-glycosylation are double-underlined. A polyadenylation signal (AT
TA AA) (23) is dot-underlined. *, nucleotide position of +1. Right ordinate,
nucleotide positions and amino acid coordinates of mature ME491 antigen.
of 237 amino acids (M, 25,475) with three putative sites for Nlinked glycosylation. Computer searches (NBRF Protein Data
Bank) revealed no significant homology with known proteins.
The apparent difference in molecular size between the estimated
molecular weight (25,475) and observed value of nonglycosyllated core protein (about 20,000) (8,10) might rest in the faster
migration of Hydrophobie membrane protein in SDS-polyacryl
amide gel than marker proteins with the same molecular sizes
(30). Hydrophobicity plot analysis (31) suggests that there are
four transmembrane regions: three consecutive ones near the
amino terminus; and the remaining one near the carboxyl
terminus (Fig. 6). AH three putative A'-glycosylation sites are
located in a region between the third and the fourth transmem
brane domains. Since the region with the putative A'-glycosyl
ation sites should be located on the extracellular surface of the
plasma membrane, it is most likely that the amino terminus of
this protein lies inside the cytoplasm.
The nucleotide sequence from -3 to +4 (GCCATGG) is a
consensus sequence for initiation of translation (32). Hence,
this ATG codon is most probably the initiation codon and the
terminal methionine is removed following translation. The
ME491 antigen sequence does not include an amino terminal
signal sequence. It has been reported, however, that some
membrane proteins with multiple transmembrane domains lack
signal sequences, since the Hydrophobie domains act as an
internal signal sequence allowing integration into the plasma
membrane (33).
The following experiments show how the cDNA sequence
corresponds to the genomic DNA. A cDNA clone (nucleotide
+30 to +790) was fragmented into two portions by Pstl diges
tions and the resulting fragments were used as probes for
Southern blot analysis. The fragment closer to the 5' end
(nucleotides +30 to +622) hybridized to the 1.5- and the 1.1kilobase BamHl-EcoRl fragments (corresponding to pMel-29
and pMel-17, respectively; see Fig. 2), but not the intervening
0.4-kilobase EcoRl-EcoRl fragment of XR31 and XR33 genomic
DNAs, while the fragment closer to the 3'-end (nucleotides
+623 to +790) hybridized only to the 1.1-kilobase fragment
(data not shown). These results, together with the results shown
in Table 1, suggest that the 5' end of the ME491 gene is located
either in the 1.5-kilobase fragments or in an adjoining small
fragment at the very end of XR31 DNA and that the intervening
0.4-kilobase fragment is located in an intron.
ME491 Gene Is Not Amplified in High Expressor Melanomas.
ME491 antigen expression in cultured human melanoma cells
differs greatly with different cell lines. WM98, WM35,
WM1158, and WM983-A cell lines are high expressors for
ME491 antigen as judged by the immunological rosette assay,
whereas A875 and HS294 are low expressors (data not shown).
Polyadenylated RNAs from some of the cell lines were analyzed
by Northern blot analysis using the cloned DNA subfragment
as a probe (Fig. 7). Consistent with results in the immunological
50
r
\
100
150
Amino acid position
\
200
Fig. 6. Hydrophobicity plot of predicted amino acid sequences of ME491
antigen. Hydrophobicity indices were determined as described by Kyte and Doolittle (31). / to /(, putative transmembrane regions; arwws, positions of asparagines for putative A'-glycosylation sites.
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MOLECULAR CLONING OF ME491 ANTIGEN
CO
O>
S
CM
CO
X
co
O
CO
cL^
0^
O)
a>
§ 5
CO
ta
m
co
O>
in
r^
oo
in
o> co
eo
00
05
00
CO
00
kb
285 —
23 —
9.4 —
18S —
4.4 —
2.3 —
2.0 —
12345
6
Fig. 7. Northern blot analysis of various human melanoma cell lines. Polyadenylated RNA extracted from cultured cells was separated on a 1% agarose/
formarmele gel (3 «ig/lane).After transfer to a nitrocellulose filter, the RNA was
probed with a 1.1-kilobase fragment of pMel-17. (Lane 1) WM98; (Lane 2)
HS294; (Lane 3) WM9, passage 19; (Lane 4) WM9, passage 130; (Lane 5)
WM35; (Lane 6) A875.
rosette assay, ME491 mRNA was much more abundant in
WM98 and WM35 than in HS294 or A875 cells. The presence
of equal amounts of polyadenylated RNAs was verified by
probing with a housekeeping gene (phosphoglycerate kinase
gene) (data not shown). To determine whether any significant
difference in DNA levels might be responsible for altering gene
expression, high molecular weight DNAs extracted from the
above cell lines were subjected to Southern blot analysis using
the 1.1-kilobase fragment of pMel-17 (Fig. 8). No significant
difference was observed between high expressors and low expressors in terms of intensity and size of DNA fragments
corresponding to the ME491 gene. These results indicate that
there is no amplification or apparent rearrangement of the
ME491 gene. The additional weaker band(s) observed in every
human DNA tested may indicate the presence of a ME491related gene. Further studies including molecular cloning of the
related gene will clarify this matter. The different pattern of the
weaker band for WM9 DNA appears to be due to restriction
fragment length polymorphism, because DNA from peripheral
blood lymphocytes of the same patient showed a pattern iden
tical with that of WM9 melanoma DNA (data not shown).
Cross-Species Sequence Homology of the ME491 Gene. Mon
key DNA was obtained from the COS cell line (34) and mouse
DNA from Ltk" and NIH/3T3 cells (35, 36). Rat and squirrel
DNA were obtained from tissue spleen cells, and chicken DNA
was obtained from chick embryo. Southern blot analysis of
these DNA samples using the 1.1-kilobase fragment of pMe 117 was performed to detect any cross-species sequence homology. Significant hybridization was observed to DNA from mon
key, rat, mouse, and squirrel, but only negligible hybridization
was detected with chick embryo under low stringency conditions
(2x SSC-0.1% SDS at 65'C) (Fig. 9). Under high stringency
conditions (0.2x SSC-0.1% SDS at 65°C),significant hybridi
zation was observed only in human, monkey, and squirrel DNA
(data not shown).
Chromosomal Localization of ME491 Gene. To determine
which human chromosome bears the gene for the ME491
1
2345678
Fig. 8. Southern blot analysis of various melanoma cell lines. Total cellular
DNA (10 jig/lane) was digested with /tornili and separated electrophoretically
on a 1% agarose gel. After transfer to a nitrocellulose filter, DNA was probed
with the 1.1-kilobase fragment of pMel-17. (Lane 1) WM98; (Lane 2) HS294;
(Lane 3) WM9; (Lane 4) WM35; (Lane 5) A875; (Lane 6) WM1158; (Lane 7)
WM983-A; (Lane 8) WI38 human fibroblasts. kb, kilobase.
antigen, a panel of 18 DNAs derived from mouse-human hy
brids retaining defined overlapping subsets of human chromo
somes was tested for the presence of ME491 sequences by
analysis of hybrid DNA-containing Southern blots after hybrid
ization with the pMel-17 subclone. Screening of hybrid DNAs
by this method showed that ME491 sequences were present in
mouse-human hybrids retaining chromosome 12 and absent in
hybrids which had lost chromosome 12. Fig. 10 summarizes
these results.
In mapping genes to chromosome 12 in earlier experiments,
we had observed that some of the hybrid cells retained some
chromosome 12-linked genes but not others. Thus, we used
hybrid cells M44cl2S5, M44cl2S9, and GL-3a to determine
the order of some of the chromosome 12-linked genes, including
ME491. As shown in Table 2, all three hybrids retain ME491
sequences, localizing the ME491 gene between 12pl2, where
K-ras-2 maps (27), and the SW756 papillomavirus integration
site 7.16.1 at 12ql2-ql3 (24).5
The assignment of the ME491 locus to human chromosome
12 was confirmed and refined by in situ hybridization of 3Hlabeled pMel-17 plasmid DNA to metaphase chromosomes
from peripheral blood lymphocytes of a normal male. After
autoradiography, metaphase spreads were analyzed for grain
localization. About 23% of all grains were located on the long
arm of chromosome 12. Over 82% of the 12q grains were
between 12ql2 and 12ql4 with most grains at 12ql3. Fig. 11
shows a histogram depicting the silver grain distribution among
the human chromosomes. The long arm of chromosome 12
represents approximately 3.3% of the haploid genome, and our
observations that more than 19% of the pMel-17 probe hybrid
ization was localized to this region are highly significant (P <
0.001). Thus cytological hybridization localizes the ME491
gene to the region between 12ql2 and 12ql4.
5 N. Popescu and J. DiPaolo, personal communication.
2959
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1988 American Association for Cancer Research.
MOLECULAR CLONING OF ME491 ANTIGEN
C\J
CO
CM
Human Chromotomes
Hybrid
O)
(O
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CO
E
-3
o
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-5. E
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O)
co
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z
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6.6 —
Fig. 10. Presence of ME491 sequences, in a panel of mouse-human hybrids.
A summary figure showing human chromosomes (top abscissa) or chromosome
regions present in a panel of mouse-human hybrids (left ordinate). Right ordinate
column, positive or negative hybridization with the pMel-17 cloned probe rep
resenting the ME491 gene. •,hybrid named on the left contains the chromosome
indicated at the top of the column; Eil.retention of the long arm of the chromosome
indicated; D. retention of the short arm of the respective chromosome; stippling
of larger or smaller fractions of the boxes, retention of more than or less than
one arm of a chromosome; U, absence of the chromosome indicated. The column
for chromosome 12 is boldly outlined and stippled to highlight the correlation of
pattern of segregation of the pMel-17 probe with the pattern of retention of
chromosome 12.
4.4 —
•
2.3 —
2.0 —
Table 2 Segregation of chromosome 12-linked probes in hybrids retaining partial
chromosome 12
Hybrids B2 and 3c-9, which retain a complete chromosome 12, and C'll,
1
23456789
Fig. 9. Detection of DNA sequences homologous to the ME49I gene in
different species. Total cellular DNA (20 «ig/lane)was examined as described in
the legend for Fig. 8 except that washing was under less stringent conditions
(2 x SSC-0.1% SDS at 65'C instead of 0.2x SSC-0.1% SDS at 65'C). (Lane 1)
WI38 human fibroblasts; (Lane 2) COS monkey fibroblasts; (Lane 3) rat spleen
cells; (Lane 4) Ltk" mouse fibroblasts; (Lane 5) NIH/3T3 mouse fibroblasts;
(Lane 6) squirrel spleen cells, A-2; (Lane 7) squirrel spleen cells, K-28; (Lane 8)
chick embryo; (Lane 9) A875 human melanoma, kh. kilobase.
which has lost chromosome 12, are described in Fig. 10. Each of the hybrids in
Table 2 was tested for the presence of each of the chromosome 12-linked genes
as described in the legend to Fig. 10.
Chromosome 12-linked DNA probes
I2ql3B2
Hybrid
+3c-9
+M44cl2S5
-(--M44cl2S9
-GL-3a
+CI1
C812pl2—
T4
K-r<u-2
12pter
I2pl2
+
+
-
+
+
+
+
Hox
12ql2—
12qter
+
+
+
+
+
_____Insulin-likegrowth
pMel17
7.16.1
I12q22—
factor
q24++——+-
+
+
DISCUSSION
+
DNA-mediated gene transfer was used to introduce the hu
+
man gene encoding the ME491 antigen (7-9) to mouse fibroblast Ltk cells and to obtain primary and secondary transformants. The gene was then molecularly cloned, using Alii human
advanced stages of melanoma. It is also to be noted that when
repetitive sequences (16) as a probe, from a X genomic library
melanocytic cells are placed in culture, the stage specificity of
prepared with DNA from one of the secondary transformants.
the antigen expression is considerably reduced (3); normal
The identity of the cloned gene as the ME491 gene is suggested
by the following observations: (a) the cloned gene, after transmelanocytes rapidly growing in culture express the ME491
antigen (data not shown), suggesting that the cellular environ
fection to Ltk cells, mediates expression of an antigen indis
ment influences antigen expression. The regulation of activa
tinguishable by Western blot analysis from the ME491 antigen
present in melanoma cells; (b) subfragments of the cloned gene, tion and suppression of the ME491 gene, and its role, if any,
in melanoma tumor progression remain unknown. To obtain
which are free of human repetitive sequences, detect a single
mRNA in both transformant and melanoma cell lines; and (c) tools to answer these questions, we cloned the ME491 gene.
We found a large variation in ME491 antigen expression
amino acid sequences deduced from nucleotide sequences of
cDNA that were cloned using the genomic subfragment as a among human melanoma cell lines; i.e., some are high exprès
probe perfectly match the INII .-terminal amino acid chromo
sors, and others are low expressors. The difference in the degree
some of purified ME491 antigen. We therefore conclude that
of the antigen expression is not associated with gene amplifi
cation or obvious gene rearrangement, unlike the findings for
the cloned gene is the ME491 gene.
Our previous reports have indicated that expression of some oncogenes and growth factor receptors in malignant cells
ME491 antigen is tumor stage specific in melanocytic cell (37-39). Interestingly, NIH/3T3 cells transfected with the
cloned ME491 gene do not express the antigen despite the
lineages in human tissues (7, 8); the antigen is strongly ex
pressed in dysplastic nevi and radial growth phase primary
stable incorporation of the gene into the NIH/3T3 genome
(data not shown), while another mouse fibroblast cell line Ltk
melanoma both of which represent the early stages of tumor
progression, whereas it is completely negative in normal tissue
can express the antigen when transfected with the same gene
(see Table 1). It has been observed that Ltk" cells express transmelanocytes and is weaker or sometimes even negative in
2960
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1988 American Association for Cancer Research.
MOLECULAR CLONING OF ME491 ANTIGEN
cluster Hox-3 (22), and the SW756 HPV 18 integration site
(24). Furthermore, Southern blot analysis revealed a possible
M E491-related gene which hybridizes weakly to the cloned
gene. Characterization of this ME491-related gene and com
parison between the two genes should provide more information
on this particular MAA.
2013105III
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ACKNOWLEDGMENTS
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We are grateful to D. M. Jackson for technical assistance, Dr. A.
Sehgal for help in preparing primary transfectant MES, and Dr. A.
Linnenbach for helpful discussions. Thanks are also due to Dr. M.
Colombo for providing rat and squirrel DNA and Dr. D. Ewert for
providing chicken DNA. We also thank the Editorial Services Depart
ment of The Wistar Institute for preparing the manuscript.
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REFERENCES
ti>
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q qi q P q pq pq
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CHROMOSOMES
Fig. 11. Localization of ME491 gene in the human genome by in situ hybrid
ization analysis. The diagram shows the grain distribution in 108 metaphases.
Abscissa, chromosomes in their relative size proportion; ordinate, number of silver
grains. The distribution of 198 grains on 108 spreads was scored; 38 were found
over 12ql2—12ql4.
acting factors which fully activate the polyoma virus enhancer
sequence and that NIH/3T3 cells lack these factors (40). Thus,
a /ra/«-acting factor(s) might be responsible for ME491 gene
activation.
The observation that the ME491 sequence is conserved
among different species suggests that this antigen has an im
portant biological function. Nucleotide sequence analysis has
suggested that ME491 antigen is a membrane-bound protein
with four transmembrane regions. No structural homology was
found with known proteins, suggesting that this is a novel
protein. Although we do not yet know the biological function
of ME491 antigen, it is significant that this antigen is strongly
expressed in the early stages of tumor progression of melanoma.
We were unable to test the oncogenic function of this gene in
NIH/3T3 cells because of the lack of expression. On the other
hand, the ME491 gene may serve as a rapid growth-inhibitory
gene. This idea comes from the observations that decrease or
disappearance of ME491 expression in melanoma in vivo co
incides with tumor progression towards more malignant stages
(7, 8) and that dysplastic nevi and radial growth phase mela
noma, both of which strongly express this antigen in vivo, hardly
grow in vitro in contrast to other stages of melanocytic cells
(3). It will be important to know if this antigen plays a role in
either progression or suppression of melanoma. We are cur
rently preparing an expression system for this antigen under
the control of a strong viral promoter and enhancer, which will
enable us to study any stimulatory or inhibitory effects of
ME491 antigen on the functions of other oncogenes in an NIH/
3T3 transformation assay.
It will also be of interest to determine any relationship
between the ME491 gene, which maps to 12ql2—»12ql3,and
other genes located in the same region of chromosome 12 such
as glioma-associated gene gli (41), int \ (42), horneo box gene
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Molecular Cloning and Characterization of an Antigen
Associated with Early Stages of Melanoma Tumor Progression
Hak Hotta, Alonzo H. Ross, Kay Huebner, et al.
Cancer Res 1988;48:2955-2962.
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