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Expression of Human Glandular Kallikrein, Hk2, in Mammalian Cells

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Expression of Human Glandular Kallikrein, Hk2, in Mammalian Cells [CANCERRESEARCH56.5397-5402.December1,1996] Expression of Human Glandular Kallikrein, hK2, in Mammalian Cells Abhay Kumar, Amita S. Goel, Timothy M. Hill, Stephen D. Mikolajczyk, Lisa S. Millar, Kristine Kuus-Reichel, and MohammadS. Saedi' Hybritech Incorporated, San Diego. California 92196-9006 ABSTRACT regulated by androgens (12—14),and share 78% an sequence homol ogy (6, 15). Because of similarities between hK2 and PSA molecules, The human kallikrein family consists of three members, hKl, hK2, and it is speculated that measuring serum concentrations of hK2 may hK3 [prostate-specific antigen (PSA)J. PSA is a widely accepted marker enhance or complement the sensitivity of the PSA test for screening, for prostate cancer. The mRNAs for both hK2 and PSA are localized staging, and monitoring of pCa and may help distinguish between pCa predominantly to prostate epithelium and are regulated by androgens. In addition, hK2 has 78% amIno acid homology to PSA. Although similari and BPH. PSA is a chymotrypsin-like protease (16—19), whereas ties to PSA make hK2 a potential prostate cancer marker, they also protein modeling studies suggest that hK2 is a trypsin-like protease impede biochemical characterization of hK2 in those human tissues and (20, 2 1). This implies that the physiological role of hK2 in prostate is body fluids where PSA is abundant. To study the expression, biosynthesis, different from PSA. The lack of knowledge regarding the biosynthesis and processing ofhK2, recombinant hK2 was expressed in the adenovirus and protein processing of hK2 has impeded the understanding of its Induced Syrian hamster tumor cell line AV12-664 (AV12-hK2). Expres physiological role in the normal and cancerous prostate gland. sion of hK2 was analyzed by Western blots and ELISA using monoclonal Significant progress has been made in understanding the biology antibodies HK1G 464.3 [specific for prohK2 (phK2)] and HK1D 106.4 and biochemistry of PSA. PSA is postulated to liquefy semenogelin [specific for phK2 and mature hK2 (hK2)J. Western blot and ELISA and enhance male fertility (16, 17, 22). In contrast, very little is known analyses showed that phK2 was secreted into the media by AV12-hK2 cells on day 1 and was gradually converted to the mature form of hK2 by day about hK2 biology. Recently, hK2 was identified in the prostate 7. N-terminal amino acid sequencingverifiedthe Westernblot and ELISA cytosol and seminal plasma where it was uncornplexed or complexed data, This demonstrates for the first time that hK2 is secreted as phK2 and with PCI (14, 23); however, biological and biochemical properties converted to hK2 extracellularly. In addition, hK2 detected in day 4—7 have not been reported. AV12-hK2-spent media was enzymatlcally active. Recombinant hK2 was To study the biochemical and biological properties of hK2, the also expressed in human prostate carcinoma cell lines, PC3 (PC3-hK2) protein needs to be overexpressed in a heterologous system. This and DU14S (DU145-hK2), that do not express endogenous hK2 or PSA. approach provides a reliable source for hK2 that is devoid of PSA. We Shnilar to AV12-hK2 cells, both cell lines secreted phK2 that was con have previously reported expression of recombinant pphK2 in Esch verted to hK2 extracellularly. phK2 was the major form detected in the erichia coli that proved to be an invaluable immunogen (24). How spent media of PC3-hK2 cells, even after 7 days, indicating a slow con ever, to study biosynthesis, protein processing, secretion, and activity, version of phK2 to hK2. hK2 was the predominant form detected in the it is imperative to express hK2 in mammalian cells. Lovgren et al. spent media of DU145-hK2 starting on day 1, indicating the rapid con version of phK2 to hK2. In this study, we demonstrate that hK2 exists in (25) reported transient, low-level expression of hK2 in BHK-2 1 cells different forms and is secreted as phK2. phK2 is then converted to using the Semiliki Forest virus system to study cross-reactivity of enzymaticaily active hK2 extracellularly. antibodies used in immunoassays for free PSA and PSA. However, this system did not provide a stable source of hK2 which is necessary for studying the biosynthetic processing of the protein. In studies INTRODUCTION presented here, hK2 was cloned and stably expressed in Syrian ham pC a2 is the most commonly diagnosed malignancy in American ster tumor cells, AV 12, and human prostate carcinoma cell lines PC3 males, with an estimated 244,000 new cases and 40,400 deaths ex and DU14S. We demonstrate for the first time that hK2 is secreted pected in 1995, accounting for 36% and 14% of total new cases and into the spent media by mammalian cells as phK2 and converted to deaths of all cancer types, respectively (1). PSA has been widely enzymatically active hK2 extracellularly. These observations establish recognized as a reliable prognostic marker for this disease (2—4). the biosynthetic processing of hK2 and suggest that phK2 may exist Current PSA tests, however, lack the specificity to distinguish be in biological fluids and could be a useful marker for prostate cancer tween pCa and BPH, especially when serum PSA value is only and/or BPH. slighfly elevated (5). hK2 and PSA belong to the family of human glandular kallikreins. The kallikreins are a multigene family of serine MATERIALS AND METHODS proteases. Three kallikreins have been identified in humans, desig nated hKl, hK2, and hK3 (PSA; Refs. 5—8).Their genes have been Expression Vectors. A 0.8-kbDNA fragmentcodingforentirepphK2was mapped to chromosome 19 (9). hKl pancreatic/renal glandular kal PCR amplified using primers A (5'-ATATGGATCCATATGTCAGCATGT GGGACCTGGUCTCTCCA), primer B (5'-ATATGGATCCTCAGGGGTF likrein is produced primarily in the kidney, pancreas, and submandib GGCTGCGATGGT), and plasmid pVL1393 containing pphK2 cDNA (gift ular salivary gland (10). hK2 and PSA share many characteristics. from Dr. Charles Young, Mayo Clinic, Rochester, MN) as the template. The Both are primarily produced by the prostate epithelium (7, 11), are PCR products were cloned into the TA cloning vector (Invitrogen Corp., San Diego, CA) to obtain TA-hK2 and sequenced to ensure the fidelity of PCR. Received 5/13/96; accepted 10/16/96. Fig. I shows the hK2 expression vectors used in the study. pGTD-hK2 was Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage constructed by cloning the 0.8-kb hK2 insert from the TA-hK2 clone into the charges. This article must therefore be hereby marked advertisement in accordance with Bc!! site of pGT-d (gift from Dr. Brian Grinnell, Eli Lilly and Co., Imdianap 18 U.S.C. Section 1734 solely to indicate this fact. I To whom requests for reprints should be addressed, at Hybritech Incorporated. Box ohs, IN) under the control of the GBMT promoter (26). Other hK2 expression 269006, San Diego, CA 92196-9006. Phone: (619) 535-8706; Fax: (619) 457-5308; vectors, pLNS-hK2 and pLNC-hK2, were obtained by cloning the 0.8-kb insert E-mail: [email protected]. from the TA-hK2 clone into the HindIII-ClaI sites of pLNSX under the control 2 ‘@‘@abbreviations used are: pCa, prostate cancer; BPH, benign prostatic hyperplasia; of SV4O promoter and HindIH-HpaI sites of pLNCX under the control of pphK2, preprohK2; phK2, prohK2; hK2, mature hK2; PSA, prostate-specific antigen; mAb, monoclonal antibody; aa, amino acid; pNA, p-nitroanilide; ACT, al-antichymo cytomegalovirus promoter, respectively. pLNSX and pLNCX were received trypsin; PCI, protein C inhibitor; IGF, insulin-like growth factor. from Dr. A. Dusty Miller (Fred Hutchinson Cancer Center, Seattle, WA; Ref. 5397 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1996 American Association for Cancer Research. MAMMALIAN EXPRESSION OF hK2 pGTD-hK2 bodies (1 : 1000 dilution of ascites) and secondary antibodies (goat anti-mouse horseradish peroxidase, 1:500; Jackson Immumosearch Laboratories, Inc., West Grove, PA) were used to probe the blots. The immunoreactive signals were detected using the enhanced chemilumimescence(Amersham, Bucking Bcll Bcll hamshire, England) system according to the manufacturer's instructions. To measure hK2 by ELISA, microtiter plates (Becton Dickinson Labware, Lincoln Park, NJ) were coated with 50 pJ of spent media overnight at 37°C. pLNS-hK2 The wells were washed with PBS + 0.1% Tweem20, blocked with 2% BSA in PBS, and incubated for 1 h at 37°C with 50 pJ of HK1D 106.4 or HK1G @ L@—M@NEO I SV4d4@ 4@LTR1 464.3. The wells were washed again with PBS + 0.1% Tween 20 and incubated for 1 h at 37°C with 50 pA of goat anti-mouse IgG Fcy antibodies Hind III Cla I coupled to horseradish peroxidase (1:500; Jackson Immunosearch Laborato ries). The wells were washed with PBS + 0.1% Tween 20 again, incubated pLNC-hK2 with o-phemylenediamine dihydrochloride (Sigma) for 15 mm, and quenched with 4 N sulfuric acid. The colonmetric reaction was measured at A490 with an ELISA reader (model EL31O;Biotek Instruments, Inc., Burlington, VT). All samples were assayed in triplicate. Spent media from AVI2 cells transfected with vector alone were used as negative control. Purified phK2'°'217―was used Hind III Hpa I to construct the standard curve. Fig. 1. Schematic representation of hK2 expression vectors. Arrows, transcription start Assay for the Measurement of hK2 Activity. Spent media from AV12- sites. hK2 cells was incubated with 1 mM pNA-derivatized peptide substrates (H- D-Pro-Phe-Arg-pNA, 5-2302; Pharmacia Hepar, Inc., Franklin, OH) in 200 mM Tris/5 mM EDTA (pH 8.0) at 37°C. The enzymatic activity of hK2 was 27). In all of the mammalian expression vectors, the orientation of the insert determined by hydrolysis of the chromogenic substrate, leading to an increase was confirmed by DNA sequencing.
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