Testisin, a New Human Serine Proteinase Expressed by Premeiotic Testicular Germ Cells and Lost in Testicular Germ Cell Tumors1
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[CANCER RESEARCH 59, 3199–3205, July 1, 1999] Testisin, a New Human Serine Proteinase Expressed by Premeiotic Testicular Germ Cells and Lost in Testicular Germ Cell Tumors1 John D. Hooper, David L. Nicol, Joanne L. Dickinson,2 Helen J. Eyre, Anthony L. Scarman, John F. Normyle, Melanie A. Stuttgen, Meaghan L. Douglas, Kate A. Lakoski Loveland, Grant R. Sutherland, and Toni M. Antalis3 Cellular Oncology Laboratory, University of Queensland Joint Oncology Program and Queensland Institute of Medical Research, Brisbane, Queensland, 4029 [J. D. H., J. L. D., A. L. S., J. F. N., M. A. S., T. M. A.]; Department of Urology, Princess Alexandra Hospital, Woolloongabba, Queensland, 4102 [D. L. N., M. L. D.]; Centre for Medical Genetics, Department of Cytogenetics and Molecular Genetics, Women’s and Children’s Hospital, Adelaide, South Australia, 5006 [H. J. E., G. R. S.]; and Institute for Reproduction and Development, Monash Medical Center, Monash University, Clayton, Victoria 3168 [K. A. L. L.], Australia ABSTRACT testicular tumors have demonstrated loss of heterozygosity on 5q, 11p15.5, 11q13.1, 13q.3, and 16p13.3 (3), suggesting that these re- We have cloned and characterized a cDNA encoding a new human serine gions may contain candidate tumor suppressor genes. proteinase, testisin, that is abundantly expressed only in the testis and is lost Testicular germ cell maturation is dynamic, requiring cell-cell in testicular tumors. The testisin cDNA was identified by homology cloning using degenerate primers directed at conserved sequence motifs within the communication and localized cell-extracellular matrix interactions catalytic regions of serine proteinases. It is 1073 nucleotides long, including (4). Regulation of such processes involves cell surface proteolysis, 942 nucleotides of open reading frame and a 113-nucleotide 3* untranslated which is important not only for matrix remodeling but also for sequence. Northern and dot blot analyses of RNA from a range of normal regulation of growth and differentiation through activation and/or human tissues revealed a 1.4-kb mRNA species that was present only in testis, release of functionally diverse effector molecules, including cyto- which was not detected in eight of eight testicular tumors. Testisin cDNA is kines, growth factors, and cell surface receptors. Not only is charac- predicted to encode a protein of 314 amino acids, which consists of a 19-amino terization of cell surface proteolysis important for understanding germ acid (aa) signal peptide, a 22-aa proregion, and a 273-aa catalytic domain, cell maturation, but cell surface proteinases also constitute potential including a unique 17-aa COOH-terminal hydrophobic extension that is new targets for anticancer therapies. predicted to function as a membrane anchor. The deduced amino acid The serine proteinases are a large multigene family, the members of sequence of testisin shows 44% identity to prostasin and contains features that are typical of serine proteinases with trypsin-like substrate specificity. which participate in proteolytic reactions that are essential to a diverse Antipeptide antibodies directed against the testisin polypeptide detected an range of physiological and pathological processes (5). These enzymes are generally expressed as inactive zymogens; activation results in immunoreactive testisin protein of Mr 35,000–39,000 in cell lysates from COS-7 cells that were transiently transfected with testisin cDNA. Immuno- rapid molecular responses without the requirement for de novo protein staining of normal testicular tissue showed that testisin was expressed in the synthesis. The involvement of serine proteinases during the later cytoplasm and on the plasma membrane of premeiotic germ cells. No staining stages of male germ cell maturation and in fertilization has been was detected in eight of eight germ cell-derived testicular tumors. In addition, documented. The testis-specific serine proteinases human acrosin (6) the testisin gene was localized by fluorescence in situ hybridization to the and mouse TESP-1 and TESP-2 (7) are recognized as playing roles short arm of human chromosome 16 (16p13.3), a region that has been during the final stages of sperm development. Additionally, the pros- associated with allellic imbalance and loss of heterozygosity in sporadic tate epithelial cell serine proteinase, PSA,4 catalyzes the liquefaction testicular tumors. These findings demonstrate a new cell surface serine proteinase, loss of which may have a direct or indirect role in the progression of seminal coagulum (8). Plasminogen activators have been impli- of testicular tumors of germ cell origin. cated in the degradation of tight junctions in the seminiferous tubules of rat testes (9). Furthermore, as yet uncharacterized serine protein- INTRODUCTION ases are also present on sperm cells as they pass through the epidid- ymis and are necessary for the segregation of sperm surface proteins Spermatogenesis, the tightly regulated and dynamic process of male into distinct domains and the attainment of fertilization competence germ cell maturation, occurs in the testis within seminiferous tubules (10, 11). and results in the transformation of a mitotic spermatogonia into a The enzymatic properties of serine proteinases are dependent on a haploid spermatozoan (1). Testicular germ cell tumors arise from catalytic triad of His, Asp, and Ser amino acids (12), which are present immature male germ cells and may differentiate along pathways in motifs that are highly conserved among family members. We have resulting in several different histological patterns (e.g., seminoma, exploited this property in the present study by using a “homology teratoma, yolk sac tumor, and mixed germ cell tumor). Individual cloning” strategy (13–15) to identify a novel serine proteinase. Tes- tumors exhibit one or more of these histological patterns. In contrast tisin is the first serine proteinase to be identified that is expressed by to many other malignancies, underlying genetic mechanisms in tes- germ cells prior to the first meiotic division and likely functions in ticular tumorigenesis have not been substantially defined. Familial proteolytic reactions that are associated with male germ cell matura- forms of testicular carcinoma are rare (2), but studies of sporadic tion. Its loss of expression by testicular tumors of germ cell origin and the localization of the testisin gene to chromosome 16 (16p13.3), a Received 2/18/99; accepted 4/29/99. region of the genome that is subject to loss of heterozygosity and The costs of publication of this article were defrayed in part by the payment of page rearrangement in human testicular cancers, suggest a potential role for charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. testisin as a tumor suppressor in testicular cancer. 1 Supported by grants from the Queensland Cancer Fund, Brisbane, Australia, and AMRAD Operations Pty. Ltd, Melbourne, Australia. J. D. H. was supported by a John Earnshaw Scholarship from the Queensland Cancer Fund and by the Bancroft Scholarship, MATERIALS AND METHODS Queensland Institute of Medical Research. J. L. D. was supported in part by a Dora Lush Post-Graduate Biomedical Scholarship from the National Health and Medical Research Homology Cloning of Testisin cDNA. Homology cloning was performed Council of Australia. by reverse transcription-PCR using degenerate oligonucleotides directed at 2 Present address: The Eye Clinic, University of Tasmania, Hobart, Tasmania, 7000, Australia. 3 To whom requests for reprints should be addressed, at Queensland Institute of 4 The abbreviations used are: PSA, prostate-specific antigen; RACE, rapid amplifica- Medical Research, Post Office Royal Brisbane Hospital, Brisbane, 4029, Queensland, tion of cDNA ends; GST, glutathione S-transferase; FISH, fluorescence in situ hybrid- Australia. Phone: 61 7 3362 0312; Fax: 61 7 3362 0107; E-mail: [email protected]. ization. 3199 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1999 American Association for Cancer Research. A NOVEL SERINE PROTEINASE LOST IN TESTICULAR CANCER conserved regions of serine proteinases (13–15). Total RNA (5 mg), isolated Hybond-N nylon membranes (Amersham, Aylesbury, United Kingdom) as from the human cervical adenocarcinoma cell line HeLa S3 (ATCC CCL 2.2) described (16). Human multiple-tissue Northern blots and a human multiple- by the method described previously (16), was reverse transcribed at 42°C using tissue dot blot (Clontech, Palo Alto, CA) were obtained commercially. The avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) in multiple-tissue Northern blot contained 2 mg of poly(A)1 RNA per lane. The m m 1 the presence of oligo(dT)12–18 (0.25 g/ l; Pharmacia Biotech, Uppsala, dot blot contained poly(A) RNA from 50 normal adult and fetal tissues Sweden), 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 10 mM MgCl2,10mM DTT, normalized to the mRNA expression levels of eight different housekeeping 32 and 0.5 mM spermidine in a total volume of 20 ml. PCR was performed using genes and ranged from 89 to 514 ng. Blots were hybridized with [ P]dCTP- 1 ml of the reverse transcriptase reaction mixture, 500 ng of each primer, 10 labeled MscI-BamHI testisin(L) fragment (nucleotides 321–861) in Ex- 3 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, and 1–2 pressHyb (Clontech) solution at 65°C and washed to a final stringency of 0.2 units of Taq polymerase (Perkin-Elmer, Norwalk, CT). The primers were: SSC-0.1% SDS at 65°C. The dot blot was washed to a final stringency of 0.13 forward, 59-ACAGAATTCTGGGTIGTIACIGCIGCICAYTG-39; and reverse, SSC-0.5% SDS at 60°C. Blots were reprobed with b-actin cDNA or an 59-ACAGAATTCAXIGGICCICCI(C/G)(T/A)XTCICC-39 (X 5 AorG, oligonucleotide probe for 18S rRNA (16) to confirm RNA loading in each Y 5 CorT;I5 inosine). Cycling conditions were as follows: 2 cycles of 94°C lane. for 2.5 min, 35°C for 2.5 min, and 72°C for 3 min; followed by 33 cycles of Production of Affinity-purified Antipeptide Polyclonal Antibodies.