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Immunology |
Weill Medical College of Cornell University, New York, New York 10021 [D. J., Y-T. C.]; Medizinische Klinik, Hämatologie-Onkologie, Krankenhaus Nordwest, 60488 Frankfurt, Germany [J. K., E. J., A. K.]; and Ludwig Institute for Cancer Research, New York Branch, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [E. S., A. O. G., M. J. S., L. J. O., Y-T. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One major challenge confronting the analysis of autologous immune responses in breast cancer, however, is the well-recognized difficulty of establishing breast cancer cell lines as targets for immunological analysis. This is in contrast to the relative ease of establishing lines from melanoma, renal cancer, and other tumor types. For this reason, the analysis of the human T-cell response against melanoma and the molecular identification of the antigens eliciting these responses are far more advanced in melanoma (17, 18, 19) than in breast cancer.
The recent development of SEREX,3 a general method to analyze the humoral immune response of cancer patients that does not require autologous tumor cell lines, provides a powerful new way to dissect the immune response to breast cancer. Our initial application of SEREX to breast cancer led to the identification of p33ING1, encoded by a putative tumor suppressor gene in breast cancer, as an immunogenic breast cancer antigen. In addition, CT antigens, shown previously to be immunogenic antigens in other tumor types, were identified (20) . In the present study, we have continued our effort to define breast cancer antigens by SEREX. Of the panel of antigens identified, a highly restricted breast autoimmunogenic differentiation antigen, NY-BR-1, was identified and characterized.
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MATERIALS AND METHODS |
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RNA Extraction and Construction of cDNA Expression Library.
Total RNA was extracted from the BR17 breast cancer sample by conventional CsCl-guanidine thiocyanate gradient method. A cDNA library was constructed in a 
-ZAP Express vector, using a commercial cDNA library kit (Stratagene).
Immunoscreening of the cDNA Library.
The unamplified cDNA expression library was screened with the autologous serum at 1:200 dilution. The screening procedure was as described previously (21)
. Briefly, the serum was diluted 1:10, preabsorbed with phage-transfected Escherichia coli lysate, further diluted to 1:200, and incubated overnight at room temperature with the nitrocellulose membranes (Schleicher & Schuell) containing the phage plaques at a density of 4000–5000 pfu/130-mm plate. After washing, the filters were incubated with alkaline phosphatase-conjugated goat antihuman Fc
secondary antibodies, and the reactive phage plaques were visualized by incubating with 5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium.
Sequence Analysis of the Reactive Clones.
The reactive clones were subcloned, purified, and in vivo excised to pBK-CMV plasmid forms (Stratagene). Plasmid DNA was prepared by using the Wizard Miniprep DNA Purification System (Promega). The inserted DNA was evaluated by EcoRI-XbaI restriction mapping, and clones representing different cDNA inserts were sequenced. The sequencing reactions were performed by the DNA Sequencing Service at Cornell University (Ithaca, NY) using Applied Biosystems PRISM (Perkin-Elmer) automated sequencers. DNA and amino acid sequences were compared with sequences in the GenBank and the EST databases using the BLAST program. Genes identical to entries in the GenBank were classified as known genes, whereas those that shared sequence identity only to ESTs and those which have no identity in either GenBank or EST databases were designated as unknown genes.
RT-PCR.
To evaluate the mRNA expression pattern of the cloned cDNA in normal and malignant tissues, total RNA was extracted from breast cancer cell lines and tumor specimens by the conventional CsCl-guanidine thiocyanate gradient method, and normal tissue RNA was obtained commercially (Clontech). Gene-specific oligonucleotide primers were designed to amplify cDNA segments of 300–600 bp in length, with the estimated primer melting temperature in the range of 65–70°C (see Figs. 2
and 4
for specific primer sequences). All primers were synthesized commercially (Operon Technologies, Alameda, CA). RT-PCR was performed using 30 amplification cycles in a thermal cycler (Perkin-Elmer) at an annealing temperature of 60°C, and the products were analyzed by 1.5% gel electrophoresis and ethidium bromide visualization.
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Hybridization Screening of a Testicular Library.
A commercially obtained testis cDNA expression library (Stratagene) was screened using a NY-BR-1 PCR product as a probe (see Fig. 2
for primer sequences), as described in the Stratagene manual. Briefly, a total of 5 x 104 pfu/150-mm plate were transferred to nitrocellulose membranes (Schleicher & Schuell), the membranes were submerged in denaturation solution (1.5 M NaCl and 0.5 M NaOH) for 5 min, transferred into neutralization solution (1.5 M NaCl and 0.5 M Tris-HCl) for 5 min, and then rinsed in 0.2 M Tris-HCl and 2x SSC. The membranes were hybridized to a 32P-labeled DNA probe at high stringency condition (68°C, aqueous buffer) and washed at high stringency condition. Positive clones were subcloned, purified, and in vivo excised to pBK-CMV plasmid forms as described above.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Four clones were derived from the two known genes, PBK-1 (BR17-76, BR17-118, and BR17-137) and TI-227 (BR17-100). PBK-1 and TI-227 are universally expressed genes, because ESTs derived from these two genes have been reported in many different normal tissues. Of the remaining clones, NY-BR-4 through NY-BR-7, represented by one clone each, were also universally expressed based on comparison to EST databank entries. The six remaining clones, BR17-1a, BR17-8, BR17-35b, BR17-44a, BR17-44b, and BR17-128, were derived from the same unknown gene, NY-BR-1. Three matching cDNA sequences for NY-BR-1 were found in the EST database, two derived from breast cancer (accession numbers AI951118 and AW373574), and the third (accession number AW170035) derived from a pooled tissue source (testis, fetal lung, and B cell), suggesting a possible tissue-restricted expression of NY-BR-1 mRNA (see below).
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To complete the missing 5' cDNA sequence, a testicular library was screened using a NY-BR-1 PCR product as a probe. One of the clones isolated during this screening extended the 5' sequence of NY-BR-1 1346 bp but did not provide a definite translation initiation site. On the basis of this cDNA sequence, a 5' RACE-PCR was performed, and the PCR product was cloned into the pGEMT plasmid vector and sequenced. This 5'-RACE sequence extended the cDNA sequence 1292 bp further 5', with the longest ORF starting at the ATG codon at position 100. No stop codon was found in the 99-bp 5' sequence, suggesting the possibility of additional 5' coding sequence in NY-BR-1. However, repeated 5'-RACE using different nested-primer pairs and adaptor-ligated cDNA derived from different NY-BR-1 mRNA-positive tissues (testis and breast, see below) failed to extend the 5' cDNA sequence further.
The available NY-BR-1 cDNA has a 4125-bp coding sequence and a 333-bp 3'-untranslated segment (submitted to GenBank, accession number AF269087). The predicted amino acid sequence from the possible ATG initiation codon (nucleotide position 100) is shown in Fig. 1
. Motif analysis of the amino acid sequence using PROSITE and Pfam search programs identified a bipartite nuclear localization signal motif at amino acid position 17–34, suggesting that NY-BR-1 is a nuclear protein. Five tandem ankyrin repeats were also identified, located at amino acid positions 49–81, 82–114, 115–147, 148–180, and 181–213. The presence of a bZIP site (DNA-binding site followed by leucine zipper motif) at amino acid position 1077–1104 suggests that this nuclear protein functions as a transcription factor. Of interest, three additional repetitive elements were identified located between the ankyrin repeats and the NH2-terminal bZIP DNA-binding site (Fig. 1)
. The first repetitive element, consisting of 357 nucleotides (119 amino acids), is tandemly repeated three times, spanning amino acid residues 459–815. The second repetitive sequence, consisting of repeats of 11 amino acids, is located between amino acids 224 and 300 (seven repeats). The third repetitive sequence, consisting of only two repeats of 34 amino acids each, is located between amino acids 301–334 (Fig. 1)
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NY-BR-1 expression in breast cancers and other tumors was examined. Twenty-five breast cancer samples were tested, and 21 of them (84%) were positive by RT-PCR (17 showed strong signals, and the other 4 samples showed weak to moderate signals; part of the data are shown in Fig. 2B
). Among 82 nonmammary tumor samples tested (36 melanomas, 26 non-small cell lung cancers, 6 colon cancers, 6 squamous cell carcinomas, 6 transitional cell carcinomas, and 2 leiomyosarcomas), only 2 melanomas showed NY-BR-1 expression.
The expression of NY-BR-1 in tissue culture lines was also examined in cell lines derived from breast tumor, melanoma, and small cell lung cancer. Four of six breast cancer cell lines (two showed very weak signals), four of eight melanoma lines (2 very weak), and 7 of 14 small cell lung cancer lines (2 very weak) were positive (data not shown).
Chromosomal Localization and Exon-Intron Organization of NY-BR-1.
Comparison of the NY-BR-1 sequences with the newly available working draft version of the human genome allowed the assignment of NY-BR-1 to chromosome 10p11.21–12.1, with at least three chromosome 10 clones showing sequence identity to NY-BR-1 (GenBank accession numbers AL157387, AL357148, and AC067744).
A comparison of NY-BR-1 cDNA and genomic sequences also permitted the definition of NY-BR-1 exon-intron organization. The amino acid coding region of this gene contains a basic framework of 19 structurally distinct exons, with at least two additional exons encoding 3'-untranslated sequence. The detailed exon-intron junction information is described in the GenBank entry (accession number AF269087). The six ankyrin repeats are encoded by exons 2–6. Of great interest was the finding that the 357-nucleotide repeating unit in NY-BR-1 cDNA is composed of six exons, exons 10–15. The available genomic sequences are incomplete in this region, and only one of the three copies of the 357-bp repeats in NY-BR-1 cDNA was identified. This finding suggests that the DNA segment between exons 10 and 15 were duplicated and inserted in tandem during evolution. In the isolated NY-BR-1 cDNA clones, three complete copies and one incomplete copy of such repeating units are present. Thus, the predicted exon sequences in NY-BR-1 can be expressed as exons 1–15-(10A-15A)-(10B-15B)-(10C-13C)-16–21, in which A, B, and C are inexact copies of the exon 10–15 sequences. The NY-BR-1 cDNA, therefore, is derived from a total of 37 exons; whether there are allelic differences in the copy number of this repetitive element (and thus the number of exons) in different individuals is currently unknown.
The available genomic sequence (GenBank AC067744) also allowed us to extend the 5' sequence of this gene beyond the cloned NY-BR-1 cDNA. Translation of the 5' genomic sequence using the previously assigned NY-BR-1 ORF led to the identification of a new translation initiation site 168 bp upstream to the previously predicted ATG initiation codon in NY-BR-1 cDNA (see text above and Fig. 1
). If this newly identified ATG is the true initiation site used in vivo, the NY-BR-1 polypeptide would contain 1397 amino acids, 56 residues longer than is depicted in Fig. 1
(additional NH2 terminus sequence: MEEISAAAVKVVPGPERPSPFSQLVYTSNDSYIVHSGDLRKIHKAASRGQVRKLEK).
Identification of NY-BR-1 Splice Variants.
Sequence comparison of the six SEREX-defined NY-BR-1 clones revealed that they were derived from two different splice variants. One variant contains an additional coding sequence of 111 bp (nucleotide nos. 3015–3125 of cloned NY-BR-1 cDNA, encoding amino acids 973-1009; see Fig. 1
), which is absent in the other variant. Comparison with the genomic sequence confirmed that this results from an alternate splicing event, with the longer variant incorporating part of the intron 33 into exon 34 (i.e., exon 17 of the basic exon-intron framework described above). Key structural elements predicted above in the NY-BR-1 sequence are present in both splice variants, suggesting no apparent difference in biological function or subcellular localization.
The expression of these two splice variants was evaluated using primers specific to the larger variant, as well as primers spanning the alternatively spliced exon. In the normal tissues analyzed, both variants showed strong expression in testis and breast by RT-PCR (but not in other tissues), with the larger variant being the dominant form in testis and the shorter variant dominant in breast. A selective group of 10 breast cancer samples were typed for these two splice forms, and results showed cotyping of the two variants (7 strong positive, 2 weaker positive, and 1 negative), with the shorter variant consistently being the predominant form.
Isolation of a NY-BR-1 Homologue Gene.
Screening testicular cDNA library with a NY-BR-1 probe identified a cDNA encoding a new gene with homology to the NY-BR-1 gene. This clone, 3673 bp excluding the poly(A) tail, corresponded to nucleotides 1–3481 of the NY-BR-1 and showed 62% homology. A DNA database search revealed no sequence identity to GenBank "nr" database, and the new gene has been designated NY-BR-1.1 (submitted to GenBank, accession number AF269088). ORF analysis showed an ORF from nucleotide 641 to the end of the cloned sequence, with 54% homology to the putative NY-BR-1 protein sequence. The ATG initiation codon of NY-BR-1.1 is preceded by a 640-bp 5'-untranslated region with scattered stop codons. Comparison of the available NY-BR-1 and NY-BR-1.1 amino acid sequences is shown in Fig. 3
. RT-PCR analysis for NY-BR-1.1 showed a tissue-restricted mRNA expression pattern distinct from NY-BR-1. Among six normal tissues examined, NY-BR-1.1 showed strong RT-PCR signal in testis, moderate signals in brain and breast tissues, and was negative in kidney, liver, and colon (Fig. 4A)
. Upon multiple repeated experiments, normal breast tissues showed either no or weak signals, consistently weaker than those observed in testis and often in brain, indicating a lower level of expression. NY-BR-1.1 expression was also examined in six breast cancer cell lines and 10 breast cancer specimens. One of six breast cancer cell lines was positive, in contrast to four of six for NY-BR-1. Eight of 10 breast cancer specimens were positive. In comparison with NY-BR-1 expression, 6 were positive for both, 1 was positive for NY-BR-1 only, 2 were positive for NY-BR-1.1 only, and 1 was negative for both (Fig. 4B)
. The strong expression in brain and low-level expression in normal breast and the lack of correlation in NY-BR-1 and NY-BR-1.1 expression in breast cancer lines and tissues indicate that these two gene products have a clearly distinct expression pattern.
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DISCUSSION |
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Differentiation antigens are antigens that show expression in specific cell lineage(s) or at specific stages of differentiation in a particular cell lineage(s) (32) . This category of antigens has been best studied in cells of lymphoid and hematopoietic derivation, starting with the definition of mouse cell surface differentiation antigens of lymphocytes, such as TL (33 , 34) , Thy-1 (35) , and Lyt-2 (36) . Application of hybridoma technology to the analysis of human cells resulted in the identification of a broad range of differentiation antigens, and this has led to the classification system for CD antigens (37) . Most of the initial CD antigens were restricted differentiation antigens expressed in lymphocytes and other hematopoietic cells, e.g., CD1 through CD8 primarily restricted to T cells (38) . The expression of differentiation antigens in normal cells is generally preserved in their neoplastic counterparts, and this feature has made these antigens useful markers in the immunopathological differential diagnosis of cancer. The best example of this is again in the hematopoietic/lymphocytic lineages, in which the antigenic profile of the neoplastic cells provided the foundation for the classification of leukemia/lymphoma (39) . In addition, these antigens can be targets for specific immunotherapy, and anti-CD20 antibody, recognizing a B-cell differentiation antigen, represents the first monoclonal antibody approved by the Food and Drug Administration for cancer immunotherapy (40) .
In addition to cells of hematopoietic origin, the melanocyte, a specialized cell type in the neuroectodermal lineage, has been found to express a number of well-characterized differentiation antigens, most of them associated with the melanin-synthesis pathway. Studies using polyclonal and monoclonal antibodies initially defined tyrosinase (41) , gp75 (42) , and gp100 (43) . Recent efforts to identify melanoma antigens recognized by CD8+ and CD4+ T cells also identified these antigens as T-cell targets and further expanded the list of melanocyte differentiation antigens (44, 45, 46, 47, 48) . Melan-A/MART-1, identified by transfection-based T-cell epitope cloning as a CD8+ T-cell target (48 , 49) , and Rab38 (50) , identified by SEREX analysis of melanoma, are two further examples of melanocyte differentiation antigens identified through these efforts. Similar to their hematopoietic counterparts, the melanocyte differentiation antigens have also been found useful in the clinical arena. Antibodies against gp100 and Melan-A/MART-1 are widely used to distinguish metastatic melanomas from other metastatic malignancies (51) , and melanoma vaccine trials targeting these antigens are being actively pursued (see Ref. 52 for an example).
With regard to common epithelial tissue, a wide range of gene products with differential expression have been defined, e.g., cytokeratins (53 , 54) , mucin-related antigens (55) , and hormonal receptors (56) . However, with rare exceptions, none of these are exclusively expressed in only a single epithelial cell type, such as breast epithelium. In this regard, NY-BR-1 is of considerable interest because of its highly restricted expression pattern in normal tissue, i.e., breast and testis. The production of antibody probes for NY-BR-1 is essential to confirm breast specificity at the protein and cell levels. With regard to cancer vaccine development, the restricted expression of NY-BR-1 in normal breast and breast cancer makes it a highly attractive vaccine target. However, the presence of a homologous gene, NY-BR-1.1, that is expressed in brain is cause for concern, and it will be necessary to show that T-cell reactivity to NY-BR-1 can be generated without cross-recognition of NY-BR-1.1.
The predicted protein sequence of NY-BR-1 contains a DNA-binding site and a leucine zipper motif (bZIP). The bZIP motif characterizes the superfamily of eucaryotic DNA-binding transcription factors that contain a basic region mediating sequence-specific DNA-binding, followed by a leucine zipper required for dimerization (57 , 58) . It is thus most likely that NY-BR-1 is a transcription factor. Five ankyrin repeats are also present in the NY-BR-1 protein. Ankyrin repeat proteins carry out a wide variety of biological activities and are found in both cytoplasm and nucleus. The repeat motif has been recognized in >400 proteins, including cyclin-dependent kinase inhibitors, transcriptional regulators, cytoskeletal organizers, developmental regulators, and toxins (59) . Thus, the ankyrin repeat in itself is not predictive of a specific cellular function or subcellular localization; rather, ankyrin repeats are thought to mediate a wide range of protein-protein interactions (59) . In comparison to other ankyrin repeat-containing proteins, NY-BR-1 is unique because of the other repetitive elements in its predicted protein sequence. These additional repetitive elements are not found in other sequences in the public protein databases, and their functional significance remains to be determined.
By comparing the cDNA sequence with the recently released working draft of the human genome, we were able to derive the following important information about NY-BR-1: (a) confirming the cDNA sequences of NY-BR-1 and NY-BR-1.1; (b) mapping to chromosome 10p11–12 and NY-BR-1.1tentatively to chromosome 9; (c) definition of the exon-intron structure of NY-BR-1, a complex gene with 37 exons, and correlate exon structure to repeating peptide units; and (d) completion of the NH2 terminus amino acid sequence of NY-BR-1 that had defied our multiple cloning efforts and RACE analysis. On the other hand, the cDNA sequences of NY-BR-1 and NY-BR-1.1 genes from this study will certainly help the annotation of corresponding genome sequences. The current study thus provides a clear example of valuable data that can be achieved by interaction between the human genome project and other scientific fields.
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FOOTNOTES |
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1 Supported by NIH Grant CA-68024 and by the Cancer Research Institute/Rhea (Rose Marie) Finnell Memorial Fellowship for breast cancer research. 
2 To whom requests for reprints should be addressed, at Department of Pathology, Weill Medical College, Cornell University, 1300 York Avenue, New York, NY 10021. E-mail: ytchen{at}med.cornell.edu
3 The abbreviations used are: SEREX, serological analysis of recombinant tumor cDNA expression libraries; CT, cancer testis; EST, expressed sequence tag; RT-PCR, reverse transcription-PCR; RACE, rapid amplification of cDNA ends; ORF, open reading frame; CD, cluster of differentiation; pfu, plaque-forming unit.
Received 8/25/00. Accepted 12/28/00.
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REFERENCES |
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is encoded by an amplified gene and induces an immune response in squamous cell lung carcinoma. Hum. Mol. Genet., 6: 33-39, 1997.This article has been cited by other articles:
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