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A Serine Proteinase Inhibitor Locus at 18Q21.3 Contains a Tandem

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A Serine Proteinase Inhibitor Locus at 18Q21.3 Contains a Tandem Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3147-3151, April 1995 Medical Sciences A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene (serpins/maspin/plasminogen activator inhibitor type 2) SANDRA S. SCHNEIDER, CHARLIE SCHICK, KIMBERLEE E. FISH, ERIC MILLER, JEANNETTE C. PENA, SARAH D. TRETER, STEPHANIE M. HuI, AND GARY A. SILVERMAN* Department of Pediatrics, Harvard Medical School, Children's Hospital, Boston, MA 02115 Communicated by Ruth Sager, Dana-Farber Cancer Institute, Boston, MA, December 30, 1994 (received for review November 4, 1994) ABSTRACT The squamous cell carcinoma antigen (SCCA) ance of the acidic fraction of SCCA is correlated with more is a member of the ovalbumin family of serine proteinase aggressive tumors. inhibitors (serpins). A neutral form of the protein is found in In our analysis ofchromosomal aberrations involving human normal and some malignant squamous cells, whereas an acidic chromosome band 18q21 we identified a DNA fragment, form is detected exclusively in tumor cells and in the circulation A56R (D18S86) (8), that contained a 56/57-bp match with the ofpatients with squamous cell tumors. In this report, we describe published cDNA sequence of SCCA (1). In this report, we the cloning of the SCCA gene from normal genomic DNA. showed that A56R contained exon 3 of a new gene, SCCA2. Surprisingly, two genes were found. They were tandemly arrayed This gene was 92% identical to SCCA1. SCCA1 and SCCA2 and flanked by two other closely related serpins, plasminogenI were tandemly arrayed and flanked by two other Ov-serpins, activator inhibitor type 2 (PAI2) and maspin at 18q213. The plasminogen activator inhibitor type 2 (PAI2) and maspin. genomic structure of the two genes, SCCA1 and SCCA2, was Finally, the predicted pI values and molecular weights of the highly conserved. The predicted amino acid sequences were 92% cDNAs suggested that the neutral and acidic forms of the identical and suggested that the neutral form of the protein was SCCA were encoded by SCCA1 and SCCA2, respectively.t encoded by SCCA1 and the acidic form was encoded by SCCA2. Further characterization ofthe region should determine whether MATERIALS AND METHODS the differential expression ofthe SCCA genes plays a causal role in developmnent of more aggressive squamous cell carcinomas. Genomic Cloning. yB29F7 was obtained from a human genomic yeast artificial chromosome (YAC) library (Wash- The squamous cell carcinoma antigen (SCCAl) gene is a ington University, St. Louis) as described (8). A rare-cutting member of the ovalbumin family of serine proteinase inhibitors restriction map of the clone has been reported (8). Genomic (Ov-serpins) (1, 2). The protein was isolated from a metastatic, DNA from yB29F7 was partially. digested with Mbo I and cervical SCC by Kato and Torigoe (3). By SDS/polyacrylamide ligated with the ADASHII vector (Stratagene) by using con- gel electrophoresis, the protein appears as a single band with ventional phage cloning techniques (9). Phage DNA was an Mr of -45,000 (1). However, isoelectric focusing reveals digested with Xba I and the resulting fragments were sub- significant charge heterogeneity with proteins being grouped cloned into pBluescript plasmid vectors (Stratagene). into a neutral (pI = 6.3-6.6; Mr 42,000-48,000) and an acidic DNA Sequencing. Plasmids were sequenced by standard fraction (pl = 5.9-6.2; Mr 42,000-48,000) (4). MonoclonalI dideoxynucleotide chain-termination methods (9) using the antibodies prepared against SCCA detect at least seven Sequenase version 2 sequencing kit (United States Biochemi- epitopes common to the neutral and acidic forms ofthe protein cals). DNA sequence was analyzed by using the Genetics and one unique to the acidic form (5). Computer Group software package and the BLAST program SCCA is detected in the superficial and intermediate layers (10) from the National Center for Biotechnology Information. of normal squamous epithelium (6), whereas the mRNA is DNA Blotting. Human genomic, phage, or plasmid DNAwas detected in the basal and subbasal levels (7). The clinical digested with restriction endonucleases and subjected to aga- import of SCCA has been as a circulating tumor marker for rose gel electrophoresis as described (8). DNA blots were SCC, especially those of the cervix, head and neck, lung, and hybridized at high stringency (8) with 32P-labeled probes esophagus (5). Multiple clinical studies of cervical SCC show generated by random priming or the PCR (9). The 2.5-kb that the percentage of patients with elevated circulating levels maspin cDNA was a generous gift from Ming Zhang and Ruth of SCCA increases from =12% at stage 0 to >90% at stage IV Sager (Cancer Genetics, Dana-Farber Cancer Institute, Bos- (5, 6). Levels fall after tumor resection and rise in =90% of the ton). patients with recurrent disease. Similar trends occur in the PCR. PCR assays were performed in 10-Al reactions and other types of SCC, with a maximum sensitivity of approxi- analyzed as described (8). The primer sequences for maspin mately 60% for lung, 50% for esophageal, and 55% for head (11), elastase inhibitor (EI) (12), and human placental throm- and neck tumors (5). Elevated circulating levels of SCCA are also detected in a lower percentage of patients with adeno- Abbreviations: El, elastase inhibitor; OVA, chicken ovalbumin; PAI2, carcinomas of the uterus, ovary, and lung (6). Interestingly, the plasminogen activator inhibitor type 2; PTI, placental thrombin in- hibitor; SCC, squamous cell carcinoma; SCCA, SCC antigen; serpin, neutral form of SCCA is detected in the cytoplasm of normal serine proteinase inhibitor; YAC, yeast artificial chromosome; UTR, and some malignant squamous cells, whereas the acidic form untranslated region; a1-AT, at-antitrypsin; a,-ACT, ai-antichymo- is expressed primarily in malignant cells and is the major form trypsin; a,-PI, at-proteinase inhibitor. found in the plasma of cancer patients (5). Thus, the appear- *To whom reprint requests should be addressed at: Children's Hos- pital, 300 Longwood Avenue, Enders 970, Boston, MA 02115. tThe sequences reported in this paper have been deposited in Gen- The publication costs of this article were defrayed in part by page charge Bank [accession nos. U19558 (SCCAl) and U19569 (SCCA2)] and the payment. This article must therefore be hereby marked "advertisement" in Genome Data Base [accession no. GOO-435-238 (EI, PTI, and maspin accordance with 18 U.S.C. §1734 solely to indicate this fact. primer sets)]. 3147 Downloaded by guest on September 27, 2021 3148 Medical Sciences: Schneider et alP Proc. Natl. Acad Sci. USA 92 (1995) bin inhibitor (PTI) (13) were derived from the published 1 2 3 4 5 6 7 8 9 10 cDNA sequence. kb For reverse transcriptase (RT)-PCR, 1-3 jig of total RNA was transcribed using random hexamer and avian myeloblas- - 12 tosis virus reverse transcriptase (9). First-strand cDNA was - 9 used in a 10-,ul PCR (14) with a primer set capable of discriminating SCCA2 transcripts from those of SCCAI (see D18S86 (A56R*) Fig. 5). RT-PCR products were subcloned into EcoRV- FIG. 2. Duplication of A56R (D18S86). Genomic DNA from 10 digested pBluescript vectors and sequenced as described (8). unrelated individuals (lanes 1-10) was digested with EcoRV. The resulting restriction fragments were separated by agarose gel electro- phoresis, blotted to reinforced nitrocellulose, hybridized with a 32p_ RESULTS labeled A56R probe (A56R*), and detected by autoradiography. Mapping, Cloning, and Sequencing of the SCCA Genes. Previously, we constructed a 2.5-Mb YAC contig to facilitate A GenBank search using the A56R sequence yielded a the analysis of 18q21 chromosomal aberrations (8). This contig 56/57-bp match with the cDNA sequence of the SCCAI (1). established that the Ov-serpin, PAI2, is 600 kb telomeric to the Appropriate acceptor and donor splice sites flanked the 57-bp protooncogene, BCL2 (8) (Fig. 1A). Restriction mapping of a sequence. This result suggested that A56R contained an exon YAC clone (yB29F7), which is located between these genes, of either SCCAI or a closely related element. To determine revealed two bands when the 210-bp A56R genomic marker which was the case, we constructed a phage library using (D18S86) was used as a probe. This result was not due to the yB29F7 genomic DNA. Three overlapping phage clones were partial digestion of genomic DNA or the existence of a similar isolated using A56R as the probe (Figs. 1C and 3). One clone, restriction site within the probe. The presence of two bands in 4A56R-2, contained two different Xba I fragments that hy- digests of DNA from 10 unrelated individuals suggested that bridized with A56R (Fig. 3). The Xba I fragments were the duplication was not an artifact of YAC cloning (Fig. 2). subcloned into plasmid vectors and analyzed by restriction A 18q21.3 TELp I ,/CEN PM2i !Dl8S86(A56R) BCL2 ' r ~~~~~~ ~~' ~~~I 100 k B yB29F7 ; ~~~~~ SinSc'I -'1<~~~~~~~~~~~~~~~~~~~~SIiS D18S79 Dl8S86(A56R)* 3UTmaspinI 5'exon -- ..J-50 kb C 4A56R .~~~~~~~~~~~~~~~~~~~~~~~~~~~~- A 3.1* 6.9 I 3.1 1 2.0 (A56R4 x x x x 1.4*. .- 1. 6.9 I 1I1J..L I %P.6 UR* iO8 A56R-2 x xa x 'PA56R-3 1.9-.- 1 341 6.8*%,.%p 5 5.4 10.4I W. .-r . ~~~~~~~~~~~~~~~~~~~~~~~~~. ~~~~~~~~~~~~~~~~~~~~~~~.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. D SCC antigen genes ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. I~~~~~~~~~~~~~~~~~~ .~~~~~~~~~~~~~~~~~~~~~~~~~~~1 II I E EE EE X ER S E R XERt R R EX S E EE E XE E S E R X I I I I 1 4I 711 L11 1-II I I I " I~~1 1L I n I 0 1 m I 2 3 4 5 6 7 8 11 2 3 4 5 6 7 8 lL184 kiqxeacint SCC antigen gene 1 SCC antigen gene 2 FIG.
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