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• W2048573811 abstract "Hyaluronic acid (HA), a glycosaminoglycan, regulates cell adhesion and migration. Hyaluronidase (HAase), an endoglycosidase, degrades HA into small angiogenic fragments. Using an enzyme-linked immunosorbent assay-like assay, we found increased HA levels (3–8-fold) in prostate cancer (CaP) tissues when compared with normal (NAP) and benign (BPH) tissues. The majority (∼75–80%) of HA in prostate tissues was found to exist in the free form. Primary CaP fibroblast and epithelial cells secreted 3–8-fold more HA than respective NAP and BPH cultures. Only CaP epithelial cells and established CaP lines secreted HAase and the secretion increased with tumor grade and metastasis. The pH activity profile and optimum (4.2; range 4.0–4.3) of CaP HAase was identical to the HYAL1-type HAase present in human serum and urine. Full-length HYAL1 transcript and splice variants were detected in CaP cells by reverse transcriptase-polymerase chain reaction, cloning, and sequencing. Immunoblotting confirmed secretion of a ∼60-kDa HYAL1-related protein by CaP cells. Immunohistochemistry showed minimal HA and HYAL1 staining in NAP and BPH tissues. However, a stromal and epithelial pattern of HA and HYAL1 expression was observed in CaP tissues. While high HA staining was observed in tumor-associated stroma, HYAL1 staining in tumor cells increased with tumor grade and metastasis. The gel-filtration column profiles of HA species in NAP, BPH, and CaP tissues were different. While the higher molecular mass and intermediate size HA was found in all tissues, the HA fragments were found only in CaP tissues. In particular, the high-grade CaP tissues, which showed both elevated HA and HYAL1 levels, contained angiogenic HA fragments. The stromal-epithelial HA and HYAL1 expression may promote angiogenesis in CaP and may serve as prognostic markers for CaP. Hyaluronic acid (HA), a glycosaminoglycan, regulates cell adhesion and migration. Hyaluronidase (HAase), an endoglycosidase, degrades HA into small angiogenic fragments. Using an enzyme-linked immunosorbent assay-like assay, we found increased HA levels (3–8-fold) in prostate cancer (CaP) tissues when compared with normal (NAP) and benign (BPH) tissues. The majority (∼75–80%) of HA in prostate tissues was found to exist in the free form. Primary CaP fibroblast and epithelial cells secreted 3–8-fold more HA than respective NAP and BPH cultures. Only CaP epithelial cells and established CaP lines secreted HAase and the secretion increased with tumor grade and metastasis. The pH activity profile and optimum (4.2; range 4.0–4.3) of CaP HAase was identical to the HYAL1-type HAase present in human serum and urine. Full-length HYAL1 transcript and splice variants were detected in CaP cells by reverse transcriptase-polymerase chain reaction, cloning, and sequencing. Immunoblotting confirmed secretion of a ∼60-kDa HYAL1-related protein by CaP cells. Immunohistochemistry showed minimal HA and HYAL1 staining in NAP and BPH tissues. However, a stromal and epithelial pattern of HA and HYAL1 expression was observed in CaP tissues. While high HA staining was observed in tumor-associated stroma, HYAL1 staining in tumor cells increased with tumor grade and metastasis. The gel-filtration column profiles of HA species in NAP, BPH, and CaP tissues were different. While the higher molecular mass and intermediate size HA was found in all tissues, the HA fragments were found only in CaP tissues. In particular, the high-grade CaP tissues, which showed both elevated HA and HYAL1 levels, contained angiogenic HA fragments. The stromal-epithelial HA and HYAL1 expression may promote angiogenesis in CaP and may serve as prognostic markers for CaP. The majority of newly diagnosed prostate cancer (CaP)1 patients have clinically organ-confined disease. The limited knowledge about which CaP is aggressive and likely to progress, as well as when it will recur, severely impedes individualized selection of therapy and subsequent prediction of outcome (1Pettway C.A. Tech. Urol. 1998; 4: 35-42PubMed Google Scholar). Routine biochemical (i.e. prostate-specific antigen levels) and surgical and pathologic parameters (i.e. Gleason sum, margin, and node status and seminal vesicle invasion) offer a glimpse of the biological potential of the tumor (2Veltri R.W. O'Dowd G.J. Orozco R. Miller M.C. Semin. Urol. Oncol. 1998; 16: 106-117PubMed Google Scholar, 3Kupelin P. Katcher J. Levin H. Zippe C. Klein E. Urology. 1996; 48: 249-260Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 4D'Amico A.V. Whittington R. Malkowicz S.B. Schiltz D. Schnall M. Tomaszewski J.E. Wein A.A. J. Urol. 1995; 154: 131-138Crossref PubMed Scopus (308) Google Scholar, 5Partin A.W. Pound C.R. Clemens J.Q. Epstein J.I. Walsh P.C. Urol. Clin. North Am. 1993; 20: 713-725Abstract Full Text PDF PubMed Google Scholar, 6Epstein J.I. Semin. Urol. Oncol. 1998; 16: 124-128PubMed Google Scholar, 7Murphy J.P. Partin A. Cancer. 1998; 15: 2233-2238Crossref Scopus (1) Google Scholar). However, many of the CaP patients (∼50–60%) with clinically localized disease have prostate-specific antigen levels between 4 and 10 ng/ml and a biopsy Gleason score between 6 and 7, which limits the prognostic capability of these markers (1Pettway C.A. Tech. Urol. 1998; 4: 35-42PubMed Google Scholar, 2Veltri R.W. O'Dowd G.J. Orozco R. Miller M.C. Semin. Urol. Oncol. 1998; 16: 106-117PubMed Google Scholar, 6Epstein J.I. Semin. Urol. Oncol. 1998; 16: 124-128PubMed Google Scholar). The prognosis of CaP patients can be improved if molecules that associate with the biological potential of CaP are identified (7Murphy J.P. Partin A. Cancer. 1998; 15: 2233-2238Crossref Scopus (1) Google Scholar). We have recently shown that both tumor-associated hyaluronic acid (HA) and tumor-derived hyaluronidase (HAase) possibly play a role in tumor progression. HA is a nonsulfated glycosaminoglycan made up of repeating disaccharide units, d-glucuronic acid andN-acetyl-d-glucosamine (8Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2078) Google Scholar). HA is a component of extracellular matrix and is present in various tissues and tissue fluids. It performs several functions in normal physiology. Concentration of HA is elevated in several cancers including bladder, colon, breast, and lung and Wilms' tumor (9Delpech B. Girard N. Bertrand P. Chauzy C. Delpech A. J. Intern. Med. 1997; 242: 41-48Crossref PubMed Scopus (142) Google Scholar, 10Wang C. Tammi M. Guo H. Tammi R. Am. J. Pathol. 1996; 148: 1861-1869PubMed Google Scholar, 11Knudson W. Am. J. Pathol. 1996; 148: 1721-1726PubMed Google Scholar, 12Setala L.P. Tammi M.I. Tammi R.H. Eskelin M.J. Lipponen P.R. Agren U.M. Parkkinen J. Alhava E.M. Kosma V.M. Br. J. Cancer. 1999; 79: 1133-1138Crossref PubMed Scopus (134) Google Scholar, 13Lin R.Y. Argenta P.A. Sullivan K.M. Stern R. Adzick N.S. J. Pediatr. Surg. 1995; 30: 304-308Abstract Full Text PDF PubMed Scopus (18) Google Scholar). We have previously shown that the urinary HA levels are 2.5–6.5-fold elevated in bladder cancer patients and serve as a highly sensitive and specific marker for detecting bladder cancer, regardless of the tumor grade (14Lokeshwar V.B. Öbek C. Soloway M.S. Block N.L. Cancer Res. 1996; 57 (; Correction: (1998) Cancer Res.58, 3191): 773-777Google Scholar, 15Lokeshwar V.B. Öbek C. Pham H.T. Wei D. Young M.J. Duncan R.C. Soloway M.S. Block N.L. J. Urol. 2000; 163: 348-356Crossref PubMed Scopus (175) Google Scholar). In tumor tissues, HA expands upon hydration and opens up spaces for tumor cell migration. Tumor cells migrate on HA-rich matrix that is mediated by cell surface HA receptors (e.g. CD44 and RHAMM; see Refs.16Ichikawa T. Itano N. Sawai T. Kimata K. Koganehira Y. Saida T. Taniguchi S. J. Invest. Dermatol. 1999; 113: 935-939Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 17Miyake H. Hara I. Okamoto I. Gohji K. Yamanaka K. Arakawa S. Saya H. Kamidono S. J. Urol. 1998; 160: 1562-1566Crossref PubMed Scopus (33) Google Scholar, 18Hall C.L. Turley E.A. J. Neurooncol. 1995; 26: 221-229Crossref PubMed Scopus (76) Google Scholar, 19Herrlich P. Morrison H. Sleeman J. Orian-Rousseau V. Konig H. Weg-Remers S. Potan H. Ann. N. Y. Acad. Sci. 2000; 910: 106-118Crossref PubMed Scopus (136) Google Scholar). HA may also offer tumor cells some protection against immune surveillance and chemotherapeutic agents (20Hoborth K. Meir U. Marberger M. Eur. Urol. 1992; 21: 206-210Crossref PubMed Scopus (63) Google Scholar). Small fragments of HA (3–25 disaccharide units) are angiogenic (21West D.C. Kumar S. Exp. Cell Res. 1992; 183: 179-196Crossref Scopus (309) Google Scholar). We have previously isolated such angiogenic HA fragments from the urine of high-grade bladder cancer patients and shown that these fragments induce endothelial cell proliferation (14Lokeshwar V.B. Öbek C. Soloway M.S. Block N.L. Cancer Res. 1996; 57 (; Correction: (1998) Cancer Res.58, 3191): 773-777Google Scholar). Furthermore, HA fragments of the same length also induce endothelial cell migration and lumen formation (22Banarjee S. Tool B.P. J. Cell Biol. 1992; 119: 643-652Crossref PubMed Scopus (48) Google Scholar). Recent studies from our laboratory demonstrate that angiogenic HA fragments interact with RHAMM on the surface of human endothelial cells and induce the MAP kinase pathway (23Lokeshwar V.B. Selzer M.G. J. Biol. Chem. 2000; 275: 27641-27649Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 24Zhang S. Chang M.C.Y. Zylka D. Turley S. Harrison R. Turley E.A. J. Biol. Chem. 1998; 273: 11342-11348Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Thus a regulated degradation of HA in tumor tissues may be important for both tumor metastasis and angiogenesis. HAases are a family of enzymes which degrade HA (25Roden L. Campbell P. Fraser R.E. Laurent T.C. Petroff H. Thompson J.N. Ciba Found. Symp. 1989; 143: 60-86PubMed Google Scholar). Initially termed as a “spreading factor,” the presence of HAase is crucial to the spread of bacterial infections and toxins present in bee, snake, and other venoms (26Tu A.T. Hendon R.R. Comp. Biochem. Physiol. B Comp. Biochem. 1983; 76: 377-383Crossref PubMed Scopus (23) Google Scholar, 27Pukrittayakamee S. Warrell D.A. Desakorn V. McMichael A.J. White N.J. Bunnag D. Toxicon. 1988; 26: 629-637Crossref PubMed Scopus (103) Google Scholar, 28Kemeny D.M. Dalton N. Lawrence A.J. Pearce F.L. Vernon C.A. Eur. J. Biochem. 1984; 139: 217-223Crossref PubMed Scopus (63) Google Scholar). In human, 6 HAase genes have been identified (29Gmachl M. Sagan S. Ketter S. Kreil G. FEBS Lett. 1993; 336: 545-548Crossref PubMed Scopus (124) Google Scholar, 30Lepperdinger G. Strobl B. Keril G. J. Biol. Chem. 1998; 273: 22466-22470Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 31Csóka T.B. Frost G.I. Heng H.H.Q. Scherer S.W. Mohapatra G. Stern R. Genomics. 1998; 48: 63-70Crossref PubMed Scopus (72) Google Scholar, 32Csóka A.B. Scherer S.W. Stern R. Genomics. 1999; 60: 356-361Crossref PubMed Scopus (207) Google Scholar). These genes cluster in two tightly linked triplets on human chromosomes 3p21.3 (HYAL1, HYAL2, and HYAL3) and 7q31.3 (HYAL4, PH20, and HYALP1) (32Csóka A.B. Scherer S.W. Stern R. Genomics. 1999; 60: 356-361Crossref PubMed Scopus (207) Google Scholar). Among these, HYAL1, HYAL2, and PH20 are relatively well studied at the protein level. HYAL1 gene encodes a HAase that is present in human serum, however, its cellular origin is unknown (31Csóka T.B. Frost G.I. Heng H.H.Q. Scherer S.W. Mohapatra G. Stern R. Genomics. 1998; 48: 63-70Crossref PubMed Scopus (72) Google Scholar). HYAL2 gene encodes a lysosomal HAase (30Lepperdinger G. Strobl B. Keril G. J. Biol. Chem. 1998; 273: 22466-22470Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar).PH20 gene encodes the testicular-type HAase that shows a broad (pH 3.2–9.0) pH activity profile (29Gmachl M. Sagan S. Ketter S. Kreil G. FEBS Lett. 1993; 336: 545-548Crossref PubMed Scopus (124) Google Scholar). In establishing the association of HAase to tumor biology, we initially showed that HAase levels are elevated in CaP and these levels correlate with CaP progression (i.e. metastatic > high-grade ≫ low-grade > benign prostatic hyperplasia (BPH)/normal) (33Lokeshwar V.B. Lokeshwar B.L. Pham H.T. Block N.L. Cancer Res. 1996; 56: 651-657PubMed Google Scholar). In cell culture studies, we observed that, primary explant cultures of CaP cells secrete elevated levels of HAase. The elevated levels of HAase have now been demonstrated in metastatic breast tumors and in several carcinoma lines (34Liu D. Pearlman E. Diacnou E. Guo K. Mori H. Haqqi T. Markowitz S. Wilson J. Sy M.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7832-7837Crossref PubMed Scopus (203) Google Scholar, 35Bertrand P. Girard N. Duval C. Anjou J.D. Chauzy C. Menard J.F. Delpech B. Int. J. Cancer. 1996; 73: 327-331Crossref Scopus (122) Google Scholar, 36Tamakoshi K. Kikkawa F. Maeda O. Suganuma N. Yamagata S. Yamagata T. Tomada Y. Br. J. Cancer. 1997; 75: 1807-1811Crossref PubMed Scopus (25) Google Scholar, 37Podyma K.A. Yamagata S. Sakata K. Yamagata T. Res. Commun. 1997; 241: 446-452Google Scholar, 38Victor R. Maingonnat C. Chauzy C. Bertrand P. Olivier A. Maunoury R. Gioannai J. Delpech B. C. R. Acad. Sci. III. 1997; 320: 805-810Crossref PubMed Scopus (15) Google Scholar, 39Csóka T.B. Frost G.I. Stern R. Metastasis. 1997; 17: 297-311PubMed Google Scholar, 40Madan A.K., Yu, K. Dhurandhar N. Cullinane C. Pang Y. Beech D.J. Oncol. Rep. 1999; 6: 607-609PubMed Google Scholar). However, the identity of the type of HAase expressed in most cancer tissues and cells is still unknown. In bladder cancer we observed that, elevated urinary HAase levels indicate the presence of G2 and G3 bladder cancer (41Pham H.T. Block N.L. Lokeshwar V.B. Cancer Res. 1997; 57: 778-783PubMed Google Scholar, 42Lokeshwar V.B. Soloway M.S. Block N.L. Cancer Lett. 1998; 131: 21-27Crossref PubMed Scopus (35) Google Scholar). Recently, we purified the first tumor-derived HAase from the urine of bladder cancer patients and showed its similarity to HYAL1 (43Lokeshwar V.B. Young M.J. Goudarzi G. Iida N. Yudin A.I. Cherr G.N. Selzer M.G. Cancer Res. 1999; 59: 4464-4470PubMed Google Scholar). We also observed the expression of HYAL1 at the transcript and protein levels, in invasive bladder cancer cell lines, which secrete high levels of a HAase in their conditioned media. This HAase activity has a pH optimum in the range 4.1–4.3 (43Lokeshwar V.B. Young M.J. Goudarzi G. Iida N. Yudin A.I. Cherr G.N. Selzer M.G. Cancer Res. 1999; 59: 4464-4470PubMed Google Scholar). In this study, using biochemical and molecular biology techniques, we have examined the expression of HA and HAase in prostate tissues and cell culture. Furthermore, we have been able to identify and characterize the type of HAase expressed in prostate cancer cells. In addition, we have localized these molecules in prostate tissues by immunohistochemistry. We also attempted to understand the function of the tumor-associated HA-HYAL1 system. Normal prostate (NAP) tissues from adults (21–50 years) were obtained from organ donors. Neoplastic and BPH tissues (∼1 g) were obtained from patients undergoing open prostatectomy. The tissue specimens were split and the mirror segment was fixed in formalin, embedded in paraffin, and sectioned; then hematoxylin and eosin staining evaluated the histologic grades of these tumors. In this study, we have included data from only those specimens, which were histologically confirmed as normal, benign, and malignant. Fresh or frozen (∼0.5–1 g) specimens were suspended in ice-cold homogenization buffer (5 mmHepes pH 7.2, 1 mm phenylmethylsulfonyl fluoride) and homogenized for 30 s in a tissue homogenizer. The tissue extracts were clarified by centrifugation at 40,000 × g for 30 min (14Lokeshwar V.B. Öbek C. Soloway M.S. Block N.L. Cancer Res. 1996; 57 (; Correction: (1998) Cancer Res.58, 3191): 773-777Google Scholar, 33Lokeshwar V.B. Lokeshwar B.L. Pham H.T. Block N.L. Cancer Res. 1996; 56: 651-657PubMed Google Scholar, 41Pham H.T. Block N.L. Lokeshwar V.B. Cancer Res. 1997; 57: 778-783PubMed Google Scholar). The supernatants were designated as “Hepes extracts.” The tissue pellets were re-extracted in 50 mm sodium acetate (pH 5.8), 6 m guanidine HCl, and 1 mmphenylmethylsulfonyl fluoride. Following clarification by centrifugation, the supernatants were designated as “guanidine extracts.” Both Hepes and guanidine extracts were assayed for HA and protein concentration. Primary cultures from prostate tissues were set up as described previously (33Lokeshwar V.B. Lokeshwar B.L. Pham H.T. Block N.L. Cancer Res. 1996; 56: 651-657PubMed Google Scholar). For culturing fibroblasts, collagenase-digested tissue fragments were cultured in RPMI 1640 + 10% fetal bovine serum medium. The fibroblast growth in cultures was confirmed by anti-vimentin staining. During second passage, when the fibroblast cultures became ∼60% confluent, the cultures were washed extensively in PBS and incubated in serum-free RPMI 1640 containing insulin, transferrin, and selenium (ITS solution, Life Technologies, Inc., Gaithersburg, MD). The serum-free conditioned medium (SF-CM) was collected after 2–3 days. The prostatic epithelial explant cultures were set up in a prostate epithelial cell growth medium, PrEGM (Prostate Epithelial Growth Medium, BioWhitaker/Clonetics, San Diego, CA) as described before (33Lokeshwar V.B. Lokeshwar B.L. Pham H.T. Block N.L. Cancer Res. 1996; 56: 651-657PubMed Google Scholar). PrEGM is a serum-free growth medium. The epithelial cell growth in cultures was confirmed by anti-cytokeratin staining (33Lokeshwar V.B. Lokeshwar B.L. Pham H.T. Block N.L. Cancer Res. 1996; 56: 651-657PubMed Google Scholar). The SF-CM from primary cultures was collected at second passage, 3 days after subculturing, and concentrated 10-fold. Prostate cancer cell lines DU145 and LNCaP, bladder cancer line HT1376, and human embryonic lung fibroblast (HL fibroblast; passage 11) were obtained from the American Type Culture Collection (Rockville, MD). The prostate cancer line PC3-ML was a gift from Dr. M. E. Stearns, Medical College of Pennsylvania, Philadelphia, PA, and the bladder cancer cell line 253J-Lung was kindly provided by Dr. Colin Dinney, M.D. Anderson Cancer Center, University of Texas, Houston, TX. All of these cell lines were cultured in RPMI 1640 + 10% fetal bovine serum and gentamycin. At ∼60% confluence, the cultures were washed three times in PBS and incubated in serum-free RPMI + ITS. The SF-CM from these cultures was collected after 2–3 days. Alternatively, CaP fibroblast and HL fibroblast were grown in the culture medium (i.e. RPMI 1640 + 10% fetal bovine serum and gentamycin) to 80–90% confluence and the conditioned medium was collected. This medium was designated as S-CM, since it contained 10% fetal bovine serum. The S-CM were collected from fibroblast cultures to examine HAase activity, such S-CM have been used previously to demonstrate HAase activity in fibroblast cultures at pH 3.7 (44Stair-Nawy S. Cs∴ka A.B. Stern R. Biochem. Biophys. Res. Commun. 1999; 266: 268-273Crossref PubMed Scopus (46) Google Scholar). HA levels in tissue extracts and SF-CM were measured using an ELISA-like assay originally developed by Fosang et al. (45Fosang A.J. Hey N.J. Carney S.L. Hardingham T.E. Matrix. 1990; 10: 306-313Crossref PubMed Scopus (109) Google Scholar), with modifications (14Lokeshwar V.B. Öbek C. Soloway M.S. Block N.L. Cancer Res. 1996; 57 (; Correction: (1998) Cancer Res.58, 3191): 773-777Google Scholar, 15Lokeshwar V.B. Öbek C. Pham H.T. Wei D. Young M.J. Duncan R.C. Soloway M.S. Block N.L. J. Urol. 2000; 163: 348-356Crossref PubMed Scopus (175) Google Scholar). Briefly, 96-well microtiter plates were coated with 25 μg/ml human umbilical cord HA (ICN Biomedicals, Costa Mesa, CA). The HA-coated wells were incubated with various amounts of tissue extracts or SF-CM (unconcentrated) from different cell types, in the presence of a biotinylated HA-binding protein. The HA-binding protein was isolated from bovine nasal cartilage according to the method described by Tengblad (46Tengblad A. Biochim. Biophys. Acta. 1979; 578: 281-289Crossref PubMed Scopus (224) Google Scholar), which utilizes HA affinity chromatography and trypsinization to isolate the HA binding part of the proteoglycan monomer. The purified HA-binding protein was biotinylated usingN-hydroxysuccinamido biotin (Sigma). The amount of biotinylated HA-binding protein bound to the microtiter wells was determined using an avidin-biotin detection system (Vector Laboratories, Inc., Burlingame, CA). The amount of HA present in each sample (ng/ml) was determined using a standard graph. We routinely normalize the amount of HA in biological fluids (e.g. urine) or in culture CM to total protein. Normalization of HA levels in biological fluids such as urine to total protein eliminates the influence of the hydration status of an individual on HA levels (15Lokeshwar V.B. Öbek C. Pham H.T. Wei D. Young M.J. Duncan R.C. Soloway M.S. Block N.L. J. Urol. 2000; 163: 348-356Crossref PubMed Scopus (175) Google Scholar). For each sample, 3 different amounts, each in duplicate, were tested. The results are expressed as mean ± S.E. HAase levels present in tissue extracts and SF-CM/S-CM were measured using an ELISA-like assay similar to that developed by Stern and Stern (47Stern M. Stern R. Matrix. 1992; 12: 397-403Crossref PubMed Scopus (83) Google Scholar), with modifications (15Lokeshwar V.B. Öbek C. Pham H.T. Wei D. Young M.J. Duncan R.C. Soloway M.S. Block N.L. J. Urol. 2000; 163: 348-356Crossref PubMed Scopus (175) Google Scholar, 41Pham H.T. Block N.L. Lokeshwar V.B. Cancer Res. 1997; 57: 778-783PubMed Google Scholar). Briefly, 96-well microtiter wells were coated with 200 μg/ml human umbilical cord HA. The HA-coated wells were incubated with various amounts of culture CM at 37 °C for 16 h in HAase assay buffer (0.1 m sodium formate, 0.15 m NaCl, pH 4.2, 0.2 mg/ml bovine serum albumin (BSA; ELISA-grade; Sigma). The HA remaining on the wells after incubation was determined using the same biotinylated HA-binding protein that is used in the HA-ELISA-like assay, and an avidin-biotin detection system. In the avidin biotin detection system, we do not include anti-keratan sulfate monoclonal antibody to enhance the signal and routinely normalize the amount of HAase activity (milliunits/ml) in any sample (CM, in this case) to total protein (mg/ml). We also routinely normalize the amount of HAase in biological fluids (e.g. urine) to eliminate the influence of the hydration status of an individual on HAase levels. This is especially important when determining urinary HAase levels of patients with hematuria (i.e. blood in urine; Ref. 15Lokeshwar V.B. Öbek C. Pham H.T. Wei D. Young M.J. Duncan R.C. Soloway M.S. Block N.L. J. Urol. 2000; 163: 348-356Crossref PubMed Scopus (175) Google Scholar). The pH activity profile of HAase present in various CM was determined as follows: 1) pooled serum from 3 normal adults (0.5 μl); 2) human urine (2.0 μl) collected from 4 normal individuals (3 adults: 2 females and 1 male: age 25–40 years and 1 child: 7 years); 3) CM (4 μl, 10-fold concentrated) from Du145 (SF-CM), CaP fibroblasts (established from a Gleason 7 CaP; SF-CM and S-CM), and HL fibroblast cultures (SF-CM and S-CM). The indicated amounts of various samples were added to HA-coated wells containing HAase assay buffer at different pH values (2.5–7.0). Between pH 3.5 and 5.0, the HAase activity was tested in buffers differing by 0.1 pH unit (i.e. pH 3.5, 3.6, 3.7 … 5.0). The control wells received the buffers of specified pH, identical to those added to the sample wells. In addition, 10-fold concentrated RPMI + ITS (SF-medium control) and RPMI + 10% fetal bovine serum + gentamycin (S-medium control) were also tested at different pH values. These media served as controls for SF-CM and S-CM collected from different cell types. The results are expressed as (control − sample)A 405; the control represents buffer only. A method described by Gutenhoneret al. (48Gutenhoner N.W. Pogrel M.A. Stern R. Matrix. 1992; 12: 388-396Crossref PubMed Scopus (95) Google Scholar) was used to detect the presence of HAase activity in various samples (48Gutenhoner N.W. Pogrel M.A. Stern R. Matrix. 1992; 12: 388-396Crossref PubMed Scopus (95) Google Scholar). Aliquots (1.2 ml) of SF-CM and S-CM were collected from DU145, CaP fibroblast, and HL fibroblast cultures. These CM and S-medium control were concentrated ∼10-fold (100 μl). A 20-μl aliquot of each concentrated SF-CM, S-CM, S-medium control, human serum (1.5 μl), 20 μl of 10-fold concentrated normal human urine and ELISA-grade BSA (10 μg) were separated on an 8.5% SDS-polyacrylamide gel containing 0.1 mg/ml HA. Four such gels were prepared and simultaneously electrophoresed. Following electrophoresis, the gels were soaked in 3% Triton X-100, to renature the HAase present in various samples. Each gel was then incubated in HAase assay buffer of pH 3.0, 3.7, 4.2, or 4.5 without BSA. Following incubation at 37 °C for 16 h, the gels were stained sequentially with 0.5% Alcian blue and 0.15% Coomassie Blue solutions and then destained. Total RNA was extracted from CaP cell lines, a Gleason 7 CaP primary epithelial explant culture and bladder cancer lines, HT1376 and 253J-Lung using a RNA extraction kit (Quiagen, Valencia, CA). Total RNA (1 μg) was subjected to first strand cDNA synthesis using the SuperscriptTM preamplification system and oligo(dT) primers (Life Technologies, Inc., Gaithersburg, MD). The cDNA was amplified using three different HYAL1-specific primer pairs. The primers were designed based on the HYAL1 cDNA sequence deposited in the GenBankTM data base (accession number HSU03056). The sequences of the first primer pair were the following: (a) HYAL1-L1 (the sequence between nucleotides 214 and 233), 5′-CTGGTGGAAGAGACAGGAAG-3′; (b) HYAL1-R1 (the reverse complementary sequence between nucleotides 564 and 583), 5′-GGAGGCAGAGCTGAGAACAG-3′. The second primer pair was designed to amplify the entire coding region of HYAL1. The sequence of the second primer pair was the following: (a) HYAL1-L2 (the sequence between nucleotides 594 and 613), 5′-TTGTCCTCGACCAGTCCCGT-3′; (b) HYAL1-R2 (the reverse complementary sequence between nucleotides 1,906 and 1,925), 5′-ATCACCACATGCTCTTCCGC-3′. The sequence of the third primer pair was designed to amplify both the long and short forms of HYAL1 transcript. The primer sequences were the following: (a) HYAL1-L3, this sequence is between nucleotides 27,274 and 27,294 in a human cosmid clone LUCA13 from 3p21.3 (GenBankTM accession number AC002455). The cDNA clones, GenBankTM accession numbers AF173154(spliced form) and HSU03056, which contain the entire HYAL1coding sequence, lack the nucleotide base “C” present at the 5′ end in the HYAL1-L3 primer, and begin with the following T, as their first nucleotide. Therefore, the sequence between nucleotides 2 and 21 of the HYAL1-L3 primer matches with the sequence between nucleotides 1 and 20 of the cDNA sequences AF173154 and HSU03056. The sequence of the HYAL1-L3 primer is, 5′-CTTCCTCCAGGAGTCTCTGGT-3′. (b) HYAL1-R3, the reverse complementary sequence between nucleotides 247 and 267 in clone AF173154 and between nucleotides 732 and 752 in clone HSU03056. This primer sequence is, 5′-TCTCCAGGCACCACTGGGTGT-3′. The PCR conditions for HYAL1-L1/R1 primer pair were the following: (a) initial melting at 94 °C for 5 min; (b) 35 cycles of 94 °C for 1 min, 62 °C for 30 s, and 72 °C for 1 min; (c) 72 °C for 10 min. For PCR analysisTaq polymerase (Promega Corp., Madison, WI) was used. The PCR conditions for HYAL1-L2/R2 and HYAL1-L3/R3 primers were the following: (a) 95 °C for 10 min (hot start); (b) 10 cycles of 94 °C for 30 s (70–60 °C) for 30 s, i.e. annealing temperature dropping by 1 °C at each cycle, 72 °C for 1 min; (c) 25 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min; (d) 72 °C for 7 min, final extension. The PCR mixture contained 5% dimethyl sulfoxide and Ampli-TaqGoldTM (PerkinElmer Life Sciences, Wellesley, MA). Prostate epithelial cell culture SF-CM were separated on an 8.5% SDS-polyacrylamide gel, under nonreducing conditions, and then blotted onto a polyvinylidene difluoride membrane. The blotted membrane was stained with 0.15% Coomassie Blue in 30% methanol for 1 min and then destained to visualize and compare total protein profile in each lane as described previously (23Lokeshwar V.B. Selzer M.G. J. Biol. Chem. 2000; 275: 27641-27649Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). This method rules out the possibility that any differences observed in the intensity of the HYAL1 band among various samples is simply due to differences in sample loading and protein transfer (23Lokeshwar V.B. Selzer M.G. J. Biol. Chem. 2000; 275: 27641-27649Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Following visualization of the total protein profile, the blot was completely destained, rehydrated, and blocked with 3% BSA in 20 mmTris-HCl, 0.15 m NaCl, and 0.05% Tween 20. The blot was probed with 5 μg/ml anti-HYAL1 antibody at 4 °C for 16 h. The anti-HYAL1 antibody was purified as the IgG fraction using protein G-Sepharose, according to the manufacturer's protocol (Amersham Pharmacia Biotech). The blot was then washed and incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:7500 dilution; Sigma), at room temperature for 2 h. The blot was then washed and developed using an alkaline phosphatase color detection system, involving nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate substrates (Bio-Rad). To determine the specificity of the immunoblot analysis, in some exp" @default.
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• W2048573811 date "2001-04-01" @default.
• W2048573811 modified "2023-09-26" @default.
• W2048573811 title "Stromal and Epithelial Expression of Tumor Markers Hyaluronic Acid and HYAL1 Hyaluronidase in Prostate Cancer" @default.
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• W2048573811 doi "https://doi.org/10.1074/jbc.m008432200" @default.
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