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[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. 2955 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1988 American Association for Cancer Research. 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. 2956 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1988 American Association for Cancer Research. 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 2957 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1988 American Association for Cancer Research. 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. 2958 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1988 American Association for Cancer Research. 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 •?Sf CO E -3 o ^ c O £ S c Q> 3 o E C O) * -5. E I g " r O) co a » = z o rr kb 239.4 — C 0) 3 CT CO 910111213141516171819202122 O >•(0 1 D CT co 1 2345678 o E 3 co < » 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 *Vl liP i iWil l'i 1il 11iifmjà lfih p1 q P q ' 'rqlÃ-f\JI'i q345 p •i-i q p Z\ « K O ACKNOWLEDGMENTS 20 15 IO b. O i 3 ¿»uà p q ijhntt P q P 6 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. JJLU. «fP q P q IO II q 7 12 2015IOS-••.|P REFERENCES ti> q b q b q q qi q P q pq pq P q pq 13 1 14 1 15P 16ÃœJP 17 1"finii" 18 119 120 211Pq 22 X 1Y 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. 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Molecular cloning and chromosomal assignment of the human homolog of i'nf-1, a mouse gene implicated in mammary tumorigenesis. Mol. Cell Biol., 4: 2532-2534, 1984. 2962 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1988 American Association for Cancer Research. 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. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/48/11/2955 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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