854 Vol. 7, 854–860, April 2001 Clinical Cancer Research Semenogelins Are Ectopically Expressed in Small Cell Lung Carcinoma Rui G. Rodrigues, Angel Panizo-Santos, disease, sensitive methods to detect residual SCLC and its JoAnne A. Cashel, Henry C. Krutzsch, regrowth are needed (see “Molecular targets for therapy of small 3 Maria J. Merino, and David D. Roberts1 cell lung cancer,” National Cancer Institute, 1999). Unfortu- nately, relatively few circulating markers for SCLC have been Laboratory of Pathology, Division of Clinical Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892 described, and their measurement has been of limited utility in monitoring this disseminated cancer3 (9–11). Therefore, better markers are needed to detect residual disease and assess tumor ABSTRACT burden in SCLC patients. Two proteins recovered from cell surface adhesion MHS-5 is a monoclonal antibody that recognizes two complexes in a small cell lung carcinoma (SCLC) cell line polypeptides in human semen (12–14). The predominant pro- were identified as fragments of the seminal plasma proteins teins constituting semen coagulum are semenogelin I, a Mr semenogelin I and semenogelin II. Association of both pro- 50,000 protein, and semenogelin II, which exists in a nongly- teins with the adhesion complexes was induced by epidermal cosylated and glycosylated form at Mr 71,000 and Mr 76,000, growth factor. Expression of semenogelins was previously respectively (15–18). MHS-5 recognizes both of the intact forms thought to be highly specific to seminal vesicles, but Western of semenogelin I and semenogelin II, as well as several proteo- blot analysis demonstrated that semenogelin II is widely lytic fragments released during liquefaction of the gel. The expressed in SCLC cell lines and occasionally in other ma- genes for semenogelin I and semenogelin II are located 11.5 kb lignant cell lines. Although semenogelin expression is nor- apart on the p arm of chromosome 20 (19), and each gene mally restricted to males, two SCLC cell lines from female patients were also positive for semenogelin II expression. contains three exons that encode the secreted protein. Immunohistochemical analysis demonstrated diffuse expres- Histological analyses have failed to detect semenogelin sion of semenogelins in 12 of 13 SCLC tumors and focal expression in any normal human tissue other than seminal expression in a minority of lung squamous and adenocarci- vesicle epithelium (14, 20, 21). This specificity has led to the nomas. Semenogelins were secreted into the medium by use of semenogelin antibodies for forensic detection of seminal cultured SCLC cells, which suggested that these proteins fluid in sexual assault. EST-expression profiling demonstrated may be useful markers for detecting residual tumor burden that semenogelin I and semenogelin II were expressed almost or recurrence of SCLC after treatment. exclusively in cDNA libraries from normal and malignant pros- tate (see Unigene Cluster Hs; 180016 and HS; 19684) correlat- INTRODUCTION ing with the path of seminal fluid passage during ejaculation. Circulating markers have proved to be valuable for assess- However, two isolated observations from the Cancer Genome 4 ing disease progression in prostate, ovarian, and colorectal can- Anatomy Project database suggest that these genes may be cers (1–5). After treatment, these markers often provide a non- sporadically expressed in malignancies derived from other tis- invasive and sensitive assessment of residual tumor burden and sues. Sequencing of a papillary renal cell carcinoma EST library regrowth after treatment of the primary malignancy. SCLC2 (NCI CGAP Kid1, papillary renal cell carcinoma, Krizman accounts for approximately 25% of lung cancers but has the protocol 1) detected semenogelin I in 1 of 1114 clones se- lowest 5-year survival of these cancers (reviewed in Refs. 6–8). quenced. Semenogelin II was detected in 1 of 4153 clones Although SCLC initially responds well to chemotherapy, the sequenced from a colon cancer EST library (NCI CGAP Co10). cancer recurs with high frequency and is often refractory to The present study demonstrates that semenogelins are ex- further treatment. Therefore, to improve the treatment of this pressed ectopically by multiple SCLC lines derived from both male and female patients and in a minority of other lung cancer cell lines, but not in breast cancer or lymphoma cell lines. Furthermore, membrane association of the semenogelin proteins was enhanced in OH-1 SCLC cells by the addition of EGF when Received 9/12/00; revised 1/8/01; accepted 1/9/01. The costs of publication of this article were defrayed in part by the the cells attached to thrombospondin-1 peptides. Semenogelin payment of page charges. This article must therefore be hereby marked immunoreactivity was also observed in most SCLC tumors, advertisement in accordance with 18 U.S.C. Section 1734 solely to which suggests these proteins may be useful markers for SCLC. indicate this fact. 1 To whom requests for reprints should be addressed, at Building 10, Room 2A33, 10 Center Drive, MSC 1500, NIH, Bethesda, MD 20892-1500. Phone: (301) 496-6264; Fax: (301) 402-0043; E-mail: [email protected]. 2 The abbreviations used are: SCLC, small cell lung carcinoma; LC/MS, 3 Internet address: http://www.conference-cast.com/webtie/sots/lung/de- liquid chromatography/mass spectrometry; EST, expressed sequence fault.htm. tag; EGF, epidermal growth factor. 4 Internet address: http://gap.nci.nih.gov/Genes/GeneFinder. Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2001 American Association for Cancer Research. Clinical Cancer Research 855 MATERIALS AND METHODS fer buffer (20% methanol in 1ϫ Tris/glycine; Bio-Rad) at 70 Proteins and Peptides. A synthetic peptide containing V for 2.5 h and washed three times with 1ϫ Tris/glycine ␣ ␤ buffer. The membrane was blocked overnight in Dulbecco’s the thrombospondin-1 sequence that binds to the 3 1 integrin, FQGVLQNVRFVF (peptide 678) was prepared as described PBS containing 1% BSA and 0.1% Tween 20. The blot was previously (22). Semenogelin was purified from seminal plasma then incubated with biotinylated MHS-5 antibody (Humagen as described (15). Recombinant EGF was obtained from R&D Fertility Diagnostics, Charlottesville, VA) at 1:1000 for2hat Systems. 37°C followed by four washes with Dulbecco’s PBS contain- Cell Lines and Reagents. Human lung cancer (OH-1, ing 0.1% Tween (13). The membrane was then incubated NCI-N592, NCI-N417, NCI-H378, NCI-H570, NCI-H727, with streptavidin-horseradish peroxidase (Amersham Phar- NCI-H157, NCI-H520, and A549), breast carcinoma (MDA- macia Biotech) diluted 1:20,000 for 1 h. Surface proteins MB-231, MCF-7, and T47D), melanoma (C32, and A2058), and were visualized with a chemiluminescent detection kit (Am- Jurkat T lymphoma cell lines were grown in RPMI 1640 con- ersham Pharmacia Biotech). taining 10% FCS (15% FCS for OH-1 cells). Immunoprecipitation. Unlabeled MHS-5 antibody Adhesion Assays. OH-1 SCLC cells (23) were dissoci- was prebound to Protein A agarose in Dulbecco’s PBS con- ated by replacing the growth medium with 2.5 mM EDTA in taining 1% BSA and 0.1% Tween 20 for2hat4°C. Beads PBS and incubating at 37°C for 10 min. Cells were then tritu- containing the bound antibody were then incubated with 500 rated and collected by centrifugation, suspended in M199 ␮l of extracted proteins overnight at 4°C. The beads were medium, and plated on the respective thrombospondin-1 pep- washed three times with Tris-buffered saline and then were tide-coated plates (Falcon 1029). OH-1 cell adhesion to throm- eluted by heating with sample buffer (50 mM Tris, 6 M urea, bospondin-1 peptide 678 (10 ␮M) was assessed with and without 30% glycerol, 2% SDS, and bromphenol blue) at 95°C for 5 EGF (5 ng/ml). Cells were incubated for2hat37°C and then min, and separated, and transferred as described above. The aspirated and washed three times with Dulbecco’s PBS. Cy- blot was then incubated with biotinylated MHS-5 antibody toskeleton-associated adhesion complexes were isolated by de- and streptavidin-horseradish peroxidase and visualized by tergent extraction of the cells using CSK buffer [0.5% Triton chemiluminescence. ␮ LC/MS Identification of Proteins. Proteins of interest X-100, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2,20 g/ml aprotinin, 1 ␮g/ml leupeptin, 1 ␮g/ml pepstatin, 1 mM phenyl- contained in one- or two-dimensional gels were analyzed by methanesulfonyl fluoride, 10 mM PIPES (pH 6.8)] for 1 min LC/MS to determine their identity. The protein spot detected in followed by sonication for 30 s to remove cell bodies (24). The the stained gel was excised and placed in a microfuge tube. The cell surface adhesion complexes, which remained attached to the gel piece was washed with methanol/ammonium bicarbonate peptide substrates, were then extracted using 0.5 ml of 1ϫ buffer, dried in vacuo, and then treated with trypsin overnight. immunoprecipitation buffer [50 mM Tris (pH 7.2), 1% Triton The resulting peptides were extracted, separated, and analyzed X-100, 1% deoxycholate, 0.1% SDS, 150 mM NaCl, and 1 mM on a Finnigan LCQ LC/MS system. The resulting run files were phenylmethanesulfonyl fluoride]. The plates were scraped, the first analyzed using Sequest database searching software. If no recovered extracts were centrifuged, and the supernatant frac- identification resulted, then further database searching and/or de tions were collected. novo sequencing was carried out. Isoelectric Focusing/Two-Dimensional Gel Electro- Immunohistochemistry and Slide Preparation. For- phoresis. Proteins extracted from the thrombospondin-1 pep- malin-fixed, paraffin-embedded representative tissue specimens tide-associated adhesion complexes were mixed with an equal were immunostained with monoclonal antibody against MHS-5 volume of rehydration buffer (8 M urea, 1% immobilized pH (dilution 1:100). Staining was performed with the EnVision gradient buffer, and 2% 3[(3-cholamidopropyl)dimethylammo- system (DAKO).