[Urine-based diagnostics: an update on the Kiel Tumor Bank]. / Uringebundene Diagnostik: Ein Update der Kieler Tumorbank.

The heterogeneity of bladder tumors in their ability to invade and metastasize and their frequent recurrence pose a challenge for physicians who treat bladder cancer patients and for the researchers who work on bladder cancer diagnosis, recurrence, and treatment-related areas. For most new bladder cancer cases, investigation begins when patients are symptomatic (i.e., hematuria or irritative voiding). This mode of detection is often inadequate for nearly 15-30% of these new cases with high-grade bladder cancer, since the tumor is already in the invasive stage at the time of diagnosis. Bladder cancer patients are on a mandatory 3-month to 6-month surveillance schedule because bladder tumors frequently recur. The current mode of detecting bladder cancer involves cystoscopy, which is an invasive and relatively expensive procedure. Voided urine cytology, the standard noninvasive marker, is highly tumor specific and has good sensitivity for detecting high-grade tumors. However, its sensitivity for detecting low-grade tumors is low; its accuracy depends on the examiner's expertise; and it is not available everywhere. Marker systems are readily available for use in practice. Their utility remains under discussion.

Elevated tissue expression of hyaluronic acid and hyaluronidase validates the HA-HAase urine test for bladder cancer.

PURPOSE: We examined the expression of 2 bladder tumor markers, hyaluronic acid (HA) and hyaluronidase (HAase), in bladder tissues and correlated tissue staining with the inferences of the HA-HAase urine test, which detects bladder cancer. MATERIALS AND METHODS: A biotinylated HA binding protein and an antiHYAL1 antibody were used to localize HA and HYAL1 type HAase, respectively, in 83 bladder tissues. Immunoblot analysis was performed using an antiHYAL1 antibody to detect HYAL1. RESULTS: A total of 12 normal bladder tissues showed no (66%) to 1+ (34%) HA staining and 0 (83%) to 1+ (17%) HYAL1 staining. The staining intensity of HA and HYAL1 increased in 71 bladder tumor specimens on chi-square analysis (p <0.001). Grade 1 tumors demonstrated 1+ (50%) to 2+ (50%) staining for HA and 1+ to 3+ staining for HYAL1 (37%, 37% and 26%, respectively). Grades 2 and 3 tumors showed 2+ to 3+ HA (94%) and HYAL1 (79%) staining. HA was expressed in tumor associated stroma and in tumor cells, whereas only tumor cells expressed HYAL1. In bladder tumor tissues HYAL1 expression was confirmed by immunoblot analysis. In 33 of the 34 patients (97%) with bladder cancer from whom urine and tumor tissue specimens were obtained at the same time 2+ to 3+ staining of HA and/or HYAL1 in 12 and 21, respectively, constituted a positive HA-HAase urine test (kappa = 0.945). CONCLUSIONS: To our knowledge this is the first report of HA localization in bladder tissues and of HYAL1 in any normal or tumor tissue. A close correlation of elevated HA and HYAL1 levels in tumor tissues with a positive HA-HAase urine test indicates that in patients with bladder cancer tumor associated HA and HYAL1 are secreted in urine, causing the HA-HAase test to be positive.

[Hyaluronic acid and hyaluronidase. 2 new bladder carcinoma markers]. / Hyaluronsäure und Hyaluronidase. Zwei neue Blasenkarzinommarker.

The heterogeneity of bladder cancer concerning progress of recurrence is an essential characteristic of this disease. Hyaluronic acid (HA) and its degrading enzyme hyaluronidase (HAase) are intricately associated with bladder cancer angiogenesis and metastasis. Tumor-associated HA and HAase are secreted in urine. In 513 urine specimens (261 bladder cancer patients, 252 patients without bladder cancer) and 83 bladder tissue specimens (71 bladder tumors, 12 normal bladder tissues), the accuracy of HA and HAase as tumor markers was studied. Elevated urinary HA levels (> or = 500 ng/ml), indicating a positive HA test, suggest the presence of bladder cancer regardless of tumor grade. Elevated urinary HAase levels (> or = 10 mU/mg) indicate high-grade (G2/G3) bladder cancer. The combined HA-HAase urine test showed 91% sensitivity and 84% specificity to detect bladder cancer. The HA-HAase test is equally sensitive for monitoring tumor recurrence. Immunohistochemistry (IHC) staining of HA and HAase in the G1 and G2/G3 bladder cancer specimens was significantly (p < 0.001) higher than in normal bladder tissue. HA and HAase appear to be useful markers in the diagnosis of bladder cancer. When compared with other noninvasive tests, the HA-HAase urine test may be less expensive and more accurate.

Stromal and epithelial expression of tumor markers hyaluronic acid and HYAL1 hyaluronidase in prostate cancer.

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 ( approximately 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 approximately 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.

Current bladder tumor tests: does their projected utility fulfill clinical necessity?

PURPOSE: We reviewed currently available bladder cancer tests in the context of the clinical expectations of a noninvasive bladder cancer test. MATERIALS AND METHODS: We reviewed the literature on bladder cancer tests that are commercially available or have shown clinical usefulness and examined how each test compares with standard methods of bladder cancer diagnosis. RESULTS: The clinical necessity for a noninvasive test for bladder cancer is 2-fold, including the early detection of high grade bladder tumors before muscle invasion and monitoring tumor recurrence or new onset. An ideal noninvasive test should be sensitive, specific, rapid, technically simple and have low intra-assay and interassay variability. Urine cytology has high specificity but limited applicability due to its relatively low sensitivity and subjective nature. Hematuria detection by Hemastix dipstick is sensitive but not specific for detecting bladder cancer. Molecules associated with bladder tumor growth and progression may serve as a basis for designing noninvasive diagnostic tests. The Food and Drug Administration approved BTA Stat and BTA TRAK tests, which detect human complement factor H and a related protein in urine, have 60% to 80% sensitivity and 50% to 70% specificity (lower in symptomatic patients) for bladder cancer. The Food and Drug Administration approved NMP22 test, which measures the level of nuclear mitotic apparatus protein in urine, has 50% to 100% sensitivity and 60% to 90% specificity. Accu-Dx detects fibrin degradation products, fibrin and fibrinogen in urine, although this test is no longer commercially available. The Immunocyt test combines cytology with an immunofluorescence technique to improve the sensitivity of cytology for detecting low grade tumors. The telomeric repeat amplification protocol assay for telomerase in exfoliated cells has 70% to 86% sensitivity and 60% to 90% specificity for bladder cancer. However, the low stability of telomerase in urine affects its sensitivity. The hyaluronic acid and hyaluronidase (HA-HAase) test, which measures the urinary level of hyaluronic acid and hyaluronidase, has 90% to 92% sensitivity and 80% to 84% specificity for bladder cancer. Quanticyt karyometry evaluates nuclear shape and DNA content of exfoliated cells to detect bladder cancer. The list of bladder tumor markers is growing rapidly and large multicenter trials are essential to assess their usefulness. CONCLUSIONS: Although currently noninvasive bladder cancer tests cannot replace cystoscopy, some have shown a promise of being clinically useful. One or a combination of these tests-markers may prove to be a prostate specific antigen for bladder cancer provided that patients and, more importantly, clinicians accept it.

Differences in hyaluronic acid-mediated functions and signaling in arterial, microvessel, and vein-derived human endothelial cells.

Hyaluronic acid (HA), a nonsulfated glycosaminoglycan, regulates cell adhesion and migration. Small HA fragments (3-25 disaccharide units) induce neovascularization. We investigated the effect of HA and a HA fragment (10-15 disaccharide units, F1) on primary human endothelial cells (ECs). Human pulmonary ECs (HPAEC) and lung microvessel ECs (HMVEC-L) bound HA (K(d) approximately 1 and 2.3 nm, respectively) and expressed 17,780 and 16,690 HA binding sites, respectively. Both ECs showed HA-mediated cell adhesion; however, HMVEC-L was 1.5-fold better. Human umbilical vein ECs neither bound HA nor showed HA-mediated adhesion. All three ECs expressed CD44 ( approximately 110 kDa). The expression of receptor for HA-mediated motility (RHAMM) (approximately 80 kDa) was the highest in HMVEC-L, followed by HPAEC and human umbilical vein ECs. RHAMM, not CD44, bound HA in all three ECs. F1 was better than HA and stimulated a 2. 5- and 1.8-fold mitogenic response in HMVEC-L and HPAEC, respectively. Both HA and F1 induced tyrosine phosphorylation of p125(FAK), paxillin, and p42/44 ERK in HMVEC-L and HPAEC, which was blocked by an anti-RHAMM antibody. These results demonstrate that RHAMM is the functional HA receptor in primary human ECs. Heterogeneity exists among primary human ECs of different vascular origins, with respect to functional HA receptor expression and function.

HA-HAase urine test. A sensitive and specific method for detecting bladder cancer and evaluating its grade.

Hyaluronic acid and HAase are intricately associated with the biology of bladder tumor angiogenesis and metastasis. Tumor-associated HA and HAase are secreted in urine. In G2 and G3 bladder tumors, HA is degraded by HAase, resulting in the generation of angiogenic HA fragments, which, in turn, are secreted in urine. An elevated urinary HA level (> or = 500 ng/mg), indicating a positive HA test, suggests the presence of bladder cancer regardless of tumor grade. The urinary HAase levels correlate with the malignant potential of bladder cancer and are elevated (> or = 10 mU/mg) in the urine of patients with G2 and G3 bladder cancer. Combining the results from the HA and the HAase tests (the HA-HAase test) yields inferences, including the detection of bladder cancer and the evaluation of its grade. The overall 92% sensitivity of the HA-HAase test to detect bladder cancer is higher than the sensitivity of individual tests with little compromise in specificity. The HA-HAase test is equally sensitive for monitoring tumor recurrence. When compared with existing noninvasive tests, the HA-HAase test may be significantly less expensive and more accurate.

The association of elevated urinary total to sulfated glycosaminoglycan ratio and high molecular mass hyaluronic acid with interstitial cystitis.

PURPOSE: A decrease in the glycosaminoglycan (GAG) layer on the urothelium is believed to be one of the possible causes of interstitial cystitis. Consequently, GAG-like substances and hyaluronic acid (HA) have been prescribed for treating this condition. To delineate the possible role of GAG and HA in the interstitial cystitis disease process, we compared the urinary levels of total GAGs (sulfated + non-sulfated), sulfated GAGs and HA in interstitial cystitis patients and normal controls. We also examined different HA species present in the urine of interstitial cystitis patients. MATERIALS AND METHODS: The total GAG and sulfated GAG levels in urine specimens of normal individuals (n = 20) and interstitial cystitis patients (n = 25) were determined by utilizing the carbazole reaction assay and the Farndale method, respectively, and were expressed as microg./mg. creatinine. Urinary HA levels were measured by applying the HA test and were expressed as ng./mg. creatinine. Gel filtration column chromatography was used to examine the profile of urinary GAGs and HA species. RESULTS: Total urinary GAGs were 2.5 to 4-fold elevated in interstitial cystitis patients with moderate to severe symptoms (Group 2; 76.2 +/- 24.8) when compared with those in normal individuals (19.9 +/- 2.5) and patients with mild symptoms (Group 1; 30.4 +/- 5.1) (p <0.001). Three urinary GAG peaks were detected in both normal and interstitial patients. However, each GAG peak from interstitial cystitis patient urine was 3 to 5-fold higher than that from normal patient urine. The sulfated GAG levels, however, remained unchanged among normal individuals (1.4 +/- 0.22), Group 1 (2.2 +/- 0.96) and Group 2 (1.6 +/- 0.38) patients (p >0.05). Consequently, the ratio of total GAGs to sulfated GAGs was elevated 3 to 3.5-fold in Group 2 patients (49.9 +/- 13.9) in comparison to that in normal individuals (16.7 +/- 2.5) and group 1 patients (14.4 +/- 4.6) (p <0.001). Urinary HA levels were marginally elevated in Group 2 patients (821. 4 +/- 247.9) when compared with those in the normal group (337.3 +/- 106.1) and Group 1 patients (540.9 +/- 166.5). In addition, a distinct high molecular mass HA species was present only in Group 2 patients. CONCLUSIONS: The increased ratio of total GAGs to sulfated GAGs and marginally elevated HA levels in urine indicate that the GAG layer is altered in interstitial cystitis patients. However, these results are in contrast to the accepted concept that a reduction in urothelial GAGs causes interstitial cystitis. The high molecular mass HA species detected in patients with severe symptoms may play a role in the pathophysiology of this disease.

Urinary hyaluronic acid and hyaluronidase: markers for bladder cancer detection and evaluation of grade.

PURPOSE: Specific patterns of progression and frequent recurrence of bladder tumors determine the choice of treatment, frequency of surveillance, quality of life, and ultimately, patient prognosis. The prognosis would be improved if an accurate noninvasive test was available for diagnosis. Identification of markers that function in bladder cancer progression would be helpful in designing such diagnostic tests. The glycosaminoglycan, hyaluronic acid (HA), promotes tumor metastasis. Hyaluronidase (HAase), an endoglycosidase, degrades HA into small fragments that promote angiogenesis. We have previously shown that both HA and HAase are associated with bladder cancer and may function in bladder tumor angiogenesis. In this study we examined whether urinary HA and HAase levels serve as bladder cancer markers. MATERIALS AND METHODS: Among the 513 urine specimens analyzed, 261 were from transitional cell carcinoma (TCC) patients, 9 from patients with non-TCC tumors, and 243 from controls (normals, patients with other genitourinary (GU) conditions or a history of bladder cancer (HxBCa)). The urinary HA and HAase levels were measured by two ELISA-like assays that utilize a biotinylated HA binding protein for detection. These levels were normalized to total urinary protein and were expressed as ng./mg. (HA test) and mU/mg. (HAase test), respectively. RESULTS: The urinary HA levels were elevated (2.5 to 6.5 fold) in bladder cancer patients (1173.7+/-173.4; n = 261) as compared with normals (246.1+/-38.5; n = 41); GU patients (306.6+/-32.2; n = 133), and patients with a HxBCa (351.1+/-49.1; n = 69) (p <0.001). The urinary HAase levels were elevated (3 to 7 fold) in G2/G3 bladder cancer patients (26.2+/-3.2) as compared with normals (4.5+/-0.9) and patients with either GU conditions (5.8+/-1.3), HxBCa (8.2+/-2.6) or G1 tumors (9.7+/-2.5) (p <0.001). The HA test showed 83.1% sensitivity, 90.1% specificity and 86.5% accuracy in detecting bladder cancer, regardless of the tumor grade. The HAase test showed 81.5% sensitivity, 83.8% specificity and 82.9% accuracy to detect G2/G3 patients. Combining the inferences of the HA and HAase tests (HA-HAase test) resulted in detection of bladder cancer, regardless of tumor grade and stage, with higher sensitivity (91.2%) and accuracy (88.3%), and comparable specificity (84.4%). CONCLUSION: Our results show that the HA-HAase urine test is a noninvasive, highly sensitive and specific method for detecting bladder cancer and evaluating its grade.

Identification of bladder tumor-derived hyaluronidase: its similarity to HYAL1.

The glycosaminoglycan hyaluronic acid (HA) and its degrading enzyme, hyaluronidase, are intricately associated with tumor metastasis and angiogenesis. HA promotes tumor cell adhesion and migration, whereas its small fragments stimulate angiogenesis. Such small HA fragments are generated from the degradation of HA by hyaluronidase. We have previously shown (V. B. Lokeshwar et al., Cancer Res., 57: 773-777, 1997) that the HA levels are elevated in the urine and tumor tissues of bladder cancer patients regardless of the tumor grade (G). The hyaluronidase levels were found to be elevated in the urine and tumor tissues of G2 and G3 bladder cancer patients. Furthermore, angiogenic HA fragments were isolated from the urine of G2/G3 bladder cancer patients, which stimulated endothelial cell proliferation, a key event in angiogenesis. In this study, we characterized the bladder tumor-derived hyaluronidase. Analysis of hyaluronidase activity in the culture-conditioned media (CM) of 11 bladder cancer cell lines, using an ELISA-like assay and a substrate (HA)-gel technique, showed that the invasive bladder cancer cell lines secrete elevated levels of a Mr approximately 60,000 hyaluronidase. Reverse transcription-polymerase chain reaction, cloning, and sequence analyses revealed the expression of an HYAL1 transcript in bladder cancer lines. HYAL1 encodes for a hyaluronidase that is present in serum. Immunoblot analysis using an anti-HYAL1 peptide IgG confirmed the presence of a Mr approximately 60,000 HYAL1-related protein in the CM of bladder cancer cell lines, in the urine specimens from G2 and G3 bladder cancer patients, and in the partially purified preparations of bladder tumor-derived hyaluronidase. No HYAL1-related protein was detected in urine specimens from normal individuals, G1 bladder cancer patients, and patients with a history of bladder cancer but no disease at the time of testing. The bladder tumor-derived hyaluronidase present in CM and partially purified preparations was found to have maximum activity at a pH range of 4.1-4.3. The identification of bladder tumor-derived hyaluronidase should help in elucidating its role in bladder tumor progression.

Secretion of bladder tumor-derived hyaluronidase activity by invasive bladder tumor cells.

Hyaluronic acid (HA), a glycosaminoglycan, promotes tumor metastasis and its small fragments are angiogenic. These small fragments are generated by degradation of HA by hyaluronidase (HAase). We measured urinary HAase levels of 196 individuals using an ELISA-like assay. The urinary HAase levels (31.1 +/- 3.7 mU/mg) of intermediate (G2) to high-grade (G3) bladder cancer patients are five- to seven-fold elevated as compared to those of normal individuals and patients with other genitourinary conditions or low-grade (G1) bladder cancer. The increase in urinary HAase levels is due to the secretion of a tumor-derived HAase which is elevated eight-fold in G2/G3 tumor tissues. The HAase in bladder tumor tissues is secreted by tumor epithelial cells and is associated with the invasive/metastatic potential of the tumor cells.

Tumor-associated hyaluronic acid: a new sensitive and specific urine marker for bladder cancer.

Hyaluronic acid (HA), a glycosaminoglycan, is known to promote tumor cell adhesion and migration, and its small fragments stimulate angiogenesis. We compared levels of HA in the urine of normal individuals and patients with bladder cancer or other genitourinary conditions, using a sensitive ELISA-like assay. Among the 144 specimens analyzed, the urinary HA levels of bladder cancer patients with G1 (255 +/- 41.7 ng/mg), G2 (291.8 +/- 68.3 ng/mg) and G3 (428.4 +/- 67 ng/mg) tumors are 4-9-fold elevated as compared to those of normal individuals (44.7 +/- 6.2 ng/mg) and patients with other genitourinary conditions (69.5 +/- 6.8 ng/mg; P < 0.001). Urinary HA measurement by the ELISA-like assay shows a sensitivity of 91.9% and specificity of 92.8% to detect bladder cancer. Thus, urinary HA measurement is a simple, noninvasive yet highly sensitive and specific method for bladder cancer detection. The increase in urinary HA concentration is a direct correlate of the elevated tumor-associated HA levels, because the HA levels are also elevated (3-5-fold) in bladder tumor tissues (P < 0.001). The profiles of urinary HA species of normal individuals and bladder cancer patients are different. Although only the intermediate-size HA species are found in the urine of normal and low-grade bladder tumor patients, the urine of high-grade bladder cancer patients contains both the high molecular mass and the small angiogenic HA fragments. These urinary HA fragments stimulate a mitogenic response (2.4-fold) in primary human microvessel endothelial cells, suggesting that the small HA fragments may regulate tumor angiogenesis by modulating endothelial cell functions.

Tumor-derived hyaluronidase: a diagnostic urine marker for high-grade bladder cancer.

The detection of high-grade bladder tumors prior to invasion is crucial for a good prognosis. We recently found that the levels of hyaluronic acid (HA), a glycosaminoglycan, are elevated in the urine of bladder cancer patients, and small angiogenic HA fragments are present in the urine of high-grade bladder cancer patients. Hyaluronidase is an enzyme that degrades HA into small angiogenic fragments. We compared the urinary hyaluronidase levels of normal individuals and patients with bladder cancer or other genitourinary conditions, using a substrate (HA)-gel technique and an ELISA-like assay. Among the 139 specimens analyzed, the urinary hyaluronidase levels in patients with G2/G3 tumors (33.4 +/- 4.5 milliunits/mg protein) are 5-8-fold higher than those in normal individuals (4.2 +/- 1.2 milliunits/mg protein) and those in patients with G1 tumors (6.5 +/- 1.7 milliunits/mg protein) or other genitourinary conditions (7.4 +/- 1.4 milliunits/mg protein; P < 0.001). Urinary hyaluronidase measurement shows a sensitivity of 100% and a specificity of 88.8% to detect high-grade bladder (G2/G3) tumors. Thus urinary hyaluronidase measurement is a simple, noninvasive yet highly specific and sensitive method for high-grade bladder cancer detection. The increase in urinary hyaluronidase levels is due to the secretion of a tumor-associated hyaluronidase into the urine because the hyaluronidase levels in G2/G3 tumor tissues are also higher (6-7-fold) than those in normal bladder and G1 tumor tissues (P < 0.001). The bladder tumor-associated hyaluronidase activity is distinct from other hyaluronidases, has a pH optimum of 4.3, and is attributed to two proteins with molecular masses of 65 kD (p65) and 55 kD (p55).

The cell adhesion molecule, GP116, is a new CD44 variant (ex14/v10) involved in hyaluronic acid binding and endothelial cell proliferation.

In this study we have found that endothelial cells from different origins all contain a CD44-related transmembrane glycoprotein, named GP116. Using a bovine aortic endothelial cell line and a standard pulse-chase protocol, we show that GP116 is synthesized as a 52-kDa nascent polypeptide precursor (p52) which is processed to GP116 as follows, p52 --> p63/65 --> p82 --> p100 --> GP116. GP116 contains approximately 8 N- and approximately 11 O-linked oligosaccharide chains (but lacks glycosaminoglycans) and interacts directly with the cytoskeletal protein, ankyrin, both in vitro (Kd approximately 1.2 nM) and in vivo. The results of GP116 amino acid composition, reverse transcriptase-polymerase chain reaction, Southern blot, Northern blot, cloning, and sequence analyses indicate that endothelial cells express this new CD44 variant that contains an exon having significant homology with human CD44 exon 14 (ex14/v10). GP116, designated as CD44 (ex14/v10), has been shown to be a major hyaluronic acid (HA) receptor (Kd approximately 0.5-0.8 nM) responsible for cell adhesion. Most importantly, we have found that the interaction between CD44(ex14/v10) and HA or a small fragment of HA (10-15 disaccharide units) induces a mitogenic response in endothelial cells. These findings suggest that this CD44 variant plays an important role in regulating endothelial cell proliferation.

Association of elevated levels of hyaluronidase, a matrix-degrading enzyme, with prostate cancer progression.

Components of extracellular matrix and the matrix-degrading enzymes are some of the key regulators of tumor metastasis and angiogenesis. Hyaluronic acid (HA), a matrix glycosaminoglycan, is known to promote tumor cell adhesion and migration, and its small fragments are angiogenic. We have compared levels of hyaluronidase, an enzyme that degrades HA, in normal adult prostate (NAP), benign prostate hyperplasia (BPH) and prostate cancer (CaP) tissues and in conditioned media from epithelial explant cultures, using a sensitive substrate(HA)-gel assay and an ELISA-like assay. The results show a significant elevation (3-10-fold) of this enzyme in tumor tissues compared to that in NAP and BPH tissues. Furthermore, the hyaluronidase levels in tissues correlates well with the tumor grade. For example, the concentrations in a locally extended CaP lesion (191 +/- 7.9 milliunits/mg protein), and low-grade tumors (9.4 +/- 1.4 milliunits/mg protein), respectively. Among the primary epithelial explant cultures, CaP cultures secrete at least 10-fold higher levels of hyaluronidase that those secreted by NAP and BPH cultures. Furthermore, among the established prostate cancer cell lines, DU145, an androgen-unresponsive metastatic line, secretes 4-fold more hyaluronidase than LNCaP, an androgen-responsive and relatively well-differentiated cell line. We also show that prostatic hyaluronidase has an apparent M(r) approximate to 55,000, a pH optimum of 4.6, and is distinct from other well-characterized hyaluronidases.

Expression of CD44 in prostate cancer cells: association with cell proliferation and invasive potential.

High level expression of the cell surface adhesion molecule CD44 standard form and its splice variants, have been causally linked to tumor metastasis. In this study, we investigated the significance of CD44 expression in human prostatic carcinoma cells. Immunocytochemistry showed high level expression of CD44 in cells from a high grade prostate tumor, and two androgen-independent, invasive prostatic carcinoma lines, PC-3 and TSU-Pr1. Normal prostatic epithelial cells and LNCaP, a low metastatic, androgen sensitive cell line, expressed none to a very low level of CD44, although mRNA transcripts were detected in all cell lines. Immunoprecipitation detected two proteins of M(r) approximately 140 kDa and 210 kDa in PC-3, and predominantly the M(r) approximately 95 kDa protein in TSU Pr1, but none in LNCaP. Most importantly, a neutralizing antibody to CD44 inhibited cell proliferation and basement membrane invasive activity, suggesting a definitive role of CD44 in prostate tumor growth and metastasis.

Mapping the fodrin binding domain in CD45, a leukocyte membrane-associated tyrosine phosphatase.

CD45 belongs to a family of high molecular mass leukocyte glycoproteins. It contains both an intrinsic protein tyrosine phosphatase (PTPase) activity and a cytoskeleton binding site in its cytoplasmic domain. Certain cytoskeletal proteins, such as fodrin (a spectrin-like molecule), are known to play an important role in the regulation of CD45's PTPase activity. In this study we mapped the fodrin binding domain of CD45 by deleting various portions of the cytoplasmic region, followed by the expression of these truncated cDNAs using an in vitro transcription/translation system. The results of these experiments indicate that the CD45 fodrin binding domain resides between amino acids 825 and 939. Construction of a fusion protein encoding the region between amino acids 825 and 939 shows that this particular sequence itself is sufficient for fodrin binding. Further analyses indicate that the sequence (930EENKKKNRN939S) in CD45 has good sequence homology with the spectrin binding domain found in the MSP1 glycoprotein of the malarial parasite. Biochemical studies, using binding competition assays, and a synthetic peptide containing the sequence 930EENKKKNRN939S, support the conclusion that the sequence between amino acids 930 and 939 is a critical part of CD45's fodrin binding domain. Further analyses indicate that this sequence is also involved in the fodrin-induced up-regulation of CD45 PTPase activity. Therefore, we suggest that fodrin binding to this domain is required for the onset of CD45-mediated signal transduction and leukocyte activation.

Ankyrin-binding domain of CD44(GP85) is required for the expression of hyaluronic acid-mediated adhesion function.

GP85 is one of the most common hemopoietic isoforms of the cell adhesion molecule, CD44. CD44(GP85) is known to contain at least one ankyrin-binding site within its 70 aa cytoplasmic domain and to bind hyaluronic acid (HA) with its extracellular domain. In this study we have mapped the ankyrin-binding domain of CD44(GP85) by deleting various portions of the cytoplasmic region followed by expression of these truncated cDNAs in COS cells. The results of these experiments indicate that the ankyrin-binding domain resides between amino acids 305 and 355. Biochemical analyses, using competition binding assays and a synthetic peptide (NGGNGT-VEDRKPSEL) containing 15 aa between aa 305 and aa 320, support the conclusion that this region is required for ankryin binding. Furthermore, we have constructed a fusion protein in which this 15 aa sequence of CD44(GP85) is transplanted onto another transmembrane protein which does not bind ankyrin. Our results show that this fusion protein acquires the ability to bind ankyrin confirming that the sequence (306NGGNGTVEDRKPSE320L) is a critical part of the ankryin-binding domain of CD44(GP85). In addition, we have demonstrated that deletion of this 15 aa ankyrin-binding sequence from CD44(GP85) results in a drastic reduction (> or = 90%) of HA-binding and HA-mediated cell adhesion. These findings strongly suggest that ankyrin binding to the cytoplasmic domain of CD44(GP85) plays a pivotal role in regulating hyaluronic acid-mediated cell-cell and cell-extracellular matrix interactions.

Hyaluronic acid-induced lymphocyte signal transduction and HA receptor (GP85/CD44)-cytoskeleton interaction.

The purposes of this study are to characterize the binding of hyaluronic acid (HA) to mouse T lymphoma cells, to measure changes in intracellular Ca2+ after HA binding, to elucidate the interaction between the HA receptor, GP85(CD44), and ankyrin in the membrane skeleton, and finally to correlate these events with HA receptor patching/capping and cell adhesion to HA. First, we established an in vivo assay using [3H]HA to measure the binding of HA to mouse T lymphoma cells, and found that the binding of [3H]HA to these cells is readily inhibited by the addition of anti-GP85(CD44) antibody suggesting that GP85(CD44) is the HA receptor. Next, we examined various signal transducing events that occur after HA binds to its receptor on mouse T lymphoma cells. The results of these studies indicate that the concentration of intracellular Ca2+ (as measured by Fura-2 fluorescence) begins to increase within seconds, and reaches a maximal level 5 min after the addition of HA to the cells. After this increase of intracellular Ca2+, HA induces both its receptors, GP85(CD44), to form patched/capped structures, and cell adhesion to HA-coated plates. Furthermore, we have determined that GP85(CD44) binds directly and specifically to ankyrin (Kd approximately 1.94 nM) in a saturable manner; and that ankyrin is preferentially accumulated underneath the HA-induced GP85(CD44) capped structures. The Ca2+ ionophore, ionomycin, was found to stimulate HA-induced receptor capping and adhesion while EGTA (a Ca2+ chelator), nefedipine/bepridil (Ca2+ channel blockers), W-7 (a calmodulin antagonist), and cytochalasin D (a microfilament inhibitor), but not colchicine (a microtubule disrupting agent), inhibit HA-induced receptor redistribution and adhesion to HA-coated plates. These findings strongly suggest that ankyrin plays an important role in linking the HA receptor, GP85(CD44), to the membrane-associated actomyosin contractile system during hyaluronic acid-mediated lymphocyte activation.

The lymphoma transmembrane glycoprotein GP85 (CD44) is a novel guanine nucleotide-binding protein which regulates GP85 (CD44)-ankyrin interaction.

In this study, we have used photoaffinity labeling by [32P]azido-GTP as well as [32P]ADP-ribosylation by pertussis toxin (PT) and cholera toxin (CT) to identify GTP-binding proteins associated with mouse T-lymphoma plasma membranes. Our results indicate that GP85 (CD44) can be photoaffinity labeled by [32P] azido-GTP and [32P]ADP-ribosylated by both PT and CT. Using purified GP85 (CD44) obtained by Triton X-100 extraction, wheat germ agglutinin-Sepharose, and anti-GP85 (CD44) antibody affinity chromatographies, we have further characterized GP85 (CD44) as a GTP-binding protein. GP85 (CD44) is found to bind guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in a time- and dose-dependent manner with a dissociation constant of 0.83 nM. Importantly, GP85 (CD44) appears to display a GTPase activity which hydrolyzes [gamma-32P]GTP at a rate of 0.011 mol of Pi released/mol of GP85 (CD44)/min. This GTPase activity can be readily inhibited by PT- or CT-mediated ribosylation of GP85 (CD44). Most interestingly, GTP binding significantly enhances the interaction of purified GP85 (CD44) with ankyrin, whereas ADP-ribosylation of GP85 (CD44) by PT or CT inhibits the GTP-induced increase in ankyrin binding to GP85 (CD44). In addition to GP85 (CD44) being the first reported transmembrane GTP-binding protein, these results suggest that GTP plays an important role in promoting the interaction between GP85 (CD44) and its underlying membrane cytoskeleton through ankyrin.