2554 A Combination of Semiquantative Telomerase Assay and In-Cell Telomerase Activity Measurement Using Exfoliated Urothelial Cells for the Detection of Urothelial Neoplasia Kazuma Ohyashiki, M.D.1 Naoyuki Yahata, M.D.1 Junko H. Ohyashiki, M.D.1 Hiroshi Iwama, M.D.1 Shigefumi Hayashi, M.D.1 Keiko Ando, M.D.1 Taku Aizawa, M.D.2 Takaaki Ito, M.D.2 Makoto Miki, M.D.2 Yoshiro Ebihara, M.D.3 1 First Department of Internal Medicine, Tokyo Medical College, Tokyo, Japan. 2 Department of Urology, Tokyo Medical College, Tokyo, Japan. 3 Second Department of Pathology, Tokyo Medical College, Tokyo, Japan. Supported in part by a grant from the Private School Foundation, a Grant-in-Aid from the Ministry of Health and Welfare for the Second Term Comprehensive 10-Year Strategy for Cancer Control, Japan, and Kyowa Hakko Co. Ltd. (Tokyo). The authors thank Professor J. W. Shay (Department of Cell Biology and Neuroscience, The University of Texas Southwestern Medical Center, Dallas, Texas) for his critical reading of this article. Address for reprints: Kazuma Ohyashiki, M.D., Tokyo Medical College, 6 –7-1 Nishi-shinjuku, Shinjuku-ku, Tokyo 160, Japan. Received January 15, 1998; revision received May 11, 1998; accepted May 11, 1998. © 1998 American Cancer Society BACKGROUND. Telomerase is a ribonucleoprotein that synthesizes telomeres. It is detected in more than 85% of samples obtained from cancer tissues, including urologic neoplasia. The authors determined telomerase activity semiquantatively and in-cell telomerase activity in exfoliated urothelial cells obtained from urologic neoplasia specimens. The goal of this study was to provide additional information regarding a noninvasive approach to the detection of urologic neoplasia. METHODS. The authors used voided urine from 23 patients with urologic neoplasia, 2 patients with nonmalignant urologic disorders, and 10 normal individuals. Semiquantative determination of telomerase activity was performed using a fluorescence-based telomeric repeat amplification protocol (TRAP), and telomerase activity at the cellular level was determined by an in situ TRAP assay. RESULTS. The fluorescence-based TRAP assay detected urinary telomerase activity in samples from 10 of 13 patients with urologic neoplasia before treatment, whereas urinary cells obtained from 3 of 10 patients (including 1 patient with relapse) during or after treatment had detectable telomerase activity. In contrast, the in situ TRAP assay detected telomerase positive cells in samples from 11 of 13 patients before treatment and 6 of 10 patients during or after treatment. Of note was a dissociation of the results of the fluorescence-based TRAP assay and those of the in situ TRAP assay for some patients. Some patients for whom telomerase activity was not detected with the fluorescence-based TRAP assay had a low frequency of telomerase positive cells in their urine. CONCLUSIONS. A combination of semiquantative analysis and an in situ TRAP assay to detect telomerase positive cells might be a useful tool in the identification and monitoring of patients with urothelial neoplasia. Cancer 1998;83:2554 – 60. © 1998 American Cancer Society. KEYWORDS: telomerase, in situ polymerase chain reaction, fluorescence-based telomeric repeat amplification protocol, urothelial neoplasia. T elomerase is a ribonucleoprotein that synthesizes erosive telomere (TTAGGG) hexametric repeats.1,2 Telomerase activity is detected in self-renewal stem cells,3 including hematopoietic stem cells,4,5 hair follicle cells,6 and the cryptic portion of the small intestine,7 whereas normal adult somatic cells do not carry this enzyme activity. Human chromosomal ends, known as telomeres, progressively shorten with cell replication in vivo and in vitro,8 and it is believed that critical shortening of telomeres without the presence of telomerase activity results in the cessation of cell division. In somatic cells, the critical shortening of telomere length may induce cell death.9,10 Although In-Cell Telomerase Activity in Urothelial Neoplasia/Ohyashiki et al. telomeres of cancer cells might also be shortened with cell replication, up-regulation of telomerase activity may compensate for telomere length erosion and maintain cellular proliferation.9,10 It has been demonstrated that approximately 86% of primary cancer tissues carry telomerase activity,11 and therefore telomerase activity is considered a new marker for cancer cells.12,13 Telomerase activity is detected using a polymerase chain reaction (PCR)– based assay called telomeric repeat amplification protocol (TRAP).14,15 With the sensitive TRAP assay, it has been determined that most bladder carcinomas carry telomerase activity.14,16 –18 Lin et al. demonstrated that most bladder carcinomas with high telomerase activity were in an advanced stage and had deep invasion,16 whereas other researchers have reported no recognizable correlation between tumor stage and the detectability of telomerase activity,17,18 thus suggesting that telomerase reactivation might occur in the early stages of carcinogenesis. This finding further indicates that detection of telomerase activity might be useful in the early detection of bladder carcinoma. Yoshida et al. reported that natural voided urine specimens from 16 of 26 patients (62%) with bladder carcinoma contained detectable telomerase activity,18 whereas Muller et al. failed to detect telomerase activity in urine specimens; they detected telomerase activity in 73% of patients with bladder carcinoma using bladder washings by physiologic saline.17 To understand the discrepancies among the reports and to provide further insight into the early detection of urothelial neoplasia, we identified telomerase positive cells in the urinary exfoliated cells using a recently developed in situ TRAP assay.19 This technique allowed us to detect telomerase activity at the single cell level. The goal of this study was to obtain more information about the detection and noninvasive follow-up of patients with urothelial neoplasia. METHODS Samples Exfoliated cells were collected from 100 mL of freshly obtained, naturally voided urine from 23 patients with urologic neoplasia. We also used urine samples obtained from 2 patients with nonmalignant urothelial disorders (one each with renal stone and hydronephrosis). Half the urine was used to detect telomerase activity using fluorescence-based TRAP assay, and the remaining was used for in situ TRAP assay. The voided urine was collected in a centrifuge tube (50 mL) and immediately cooled on ice and centrifuged. To avoid contamination of red blood cells that interfere with PCR, the pelleted cells were resuspended in 30 mL of filtrated hypotonic 2555 solution (1.2114 g Tris, 0.7456 g KCl, and 0.2033 g MgCl26H2O to 1 liter H2O) and pipetted 15 times and then 10 mL filtrated hypertonic solution (16 g NaCl, 0.4 g KCl, 5.795 g Na2HPO4-12H2O, and 0.4 g KH2PO4 to 1 liter H2O) was added immediately.20 Half the suspended cells were centrifuged, and the cell pellet was stored at – 80°C until analysis of telomerase activity. The remaining half of the suspended cells were cytospinned (at 400 rpm for 3 minutes) on silane-coated nonfluorescence glass slides and air-dried by fan (within 5 minutes). We used COLO #320DM (the colon carcinoma cell line) as a positive control. Fluorescence-Based TRAP Assay Telomerase activity was assessed according to the method of Kim et al.,14,15 with minor modifications.21,22 After the addition of 10 –50 L telomerase assay lysis buffer (0.5% CHAPS [3-cholamidopropyldimethylammonio-1-propane-sulfate; Pierce Chemical Co., Rockford, IL], 5 mM mercaptoethanol [Sigma Chemical Co., St, Louis, MO], and 0.1 mM AEBSF [4-(2-aminothyl)-benzensulfonyl-fluoride-hydrochlorine, ICN Biomedicals, Inc., Irvine, CA]), the cells were lysed on ice. The lysate was incubated on ice for 30 minutes and then centrifuged at 10,000 relative centrifugal force for 30 minutes at 4°C. The supernatant was collected, and the protein content was determined by standard procedures (BCA protein assay). A volume of 6 g or 0.6 g protein equivalent was added to a 48 L reaction solution consisting of TRAP buffer (Oncor Inc., MD), dNTP’s mix (Oncor), TRAP primer mix (Oncor), 2 units Taq polymerase (Takara Shuzo, Shiga, Japan), and 10 pmol FITC-labeled TS primer (5´-AAT CCG TCG AGC AGA GTT-3´: 5´-end labeling using FluorePrimeTM (Pharmacia Biotech, Uppsala, Sweden). The PCR conditions were 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 15 minutes. We used a TRAP-eze detection kit (Oncor) that included 36 bp internal standard,23 FITC-labeled TS primer, and an automated laser fluorescence (ALF) DNA sequencer II (Pharmacia Biotech), and we performed analysis with the Fragment Manager program (Pharmacia Biotech).21,22 The PCR products (1.5 L) were subjected to 12% acrylamide denaturing gel electrophoresis. The telomerase activity signals in each sample were normalized to the signal from the internal standard, then expressed as a relative value. In Situ TRAP Assay In situ PCR was performed as described previously.19 Twenty-five L containing 20 mM Tris HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.05% Tween 20, 1 mM ethyleneglycoltetraacetic acid, 50 M deoxynucleoside triphosphates, 1 g of T4 gene 32 protein (Boehr- 2556 CANCER December 15, 1998 / Volume 83 / Number 12 inger Mannheim, Indianapolis, IN), bovine serum albumin (0.1 mg/mL), 2 units of Taq DNA polymerase, and 10 pmol FITC-labeled TS forward-primer (5⬘-AAT CCG TCG AGC AGA GTT-3⬘) were placed within each frame, and the slides were incubated for 30 minutes at 22°C. After TS extension, 25 L of the same solution but with 10 pmol FITC-labeled CX reverse-primer (5⬘CCC TTA CCC TTA CCC TTA CCC TAA-3⬘) were added, coverslips were sealed, and the solution was heated to 90°C for 1.5 minutes to inactivate telomerase and then amplified using a Hybrid OmniSlide System thermocycler (National Labnet Co., Woodbridge, NJ). The PCR conditions were 30 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1.5 minutes. Frames and top liners for the in situ PCR were removed completely, and slides were washed in tap water and then sealed with a cover glass using MacIlvaine buffer/ glycerin solution (1:1 ⫽ volume:volume). Cells were observed with a fluorescence microscope using a Bfilter (Nikon, Tokyo, Japan). At least 100 cells of each specimen were examined, and cells with a fluorescence positive nucleus were considered positive for telomerase activity. When a cell had brighter fluorescence in the cytoplasm than in the nucleus, the cell was considered negative for telomerase activity. Because the primers themselves were fluorescent, we performed in situ TRAP without PCR as a control, whereas other controls (data not shown) included using only one primer, PCR without primers, or PCR without Taq polymerase as previously described.19 RESULTS Fluorescence-Based TRAP Assay to Detect Telomerase Activity in Exfoliated Urothelial Cells from Urologic Neoplasia Fluorescence-based TRAP assay could detect low levels of telomerase activity semiquantatively.21,22 In the exfoliated urothelial cells, telomerase activity was detected in 10 of 13 untreated patients (relative telomerase activity ranging from 0.68 to 30.3 in the 10 patients) with various forms of urologic neoplasia, including 7 of 7 with bladder carcinoma, 1 of 2 with renal cell carcinoma, 1 of 3 with renal pelvic tumor, and 1 patient with renal metastasis of lung carcinoma (Table 1 and Fig. 1). One of the 2 patients with bladder carcinoma after surgery (Patient 11) showed telomerase activity, and 1 patient (Patient 10) with bladder carcinoma who received chemotherapy had telomerase activity in the urine (Table 1). Of the 7 patients with bladder carcinoma before treatment, 2 patients (Patients 3 and 7) had negative results when 6 g protein were applied to the CHAPS extract, but both of them had detectable telomerase activity after dilution, indicating the presence of inhibitors of PCR in the samples (Fig. 1). In voided urine from 10 normal individuals and 2 patients with nonmalignant urologic disorders (Patients 24 and 25), no telomerase activity was detectable (Table 1 and Fig. 1). Determination of In-Cell Telomerase Activity Using an In Situ TRAP Assay Six patients with bladder carcinoma before treatment had a high frequency of fluorescence positive cells (ⱖ3 cells with bright fluorescence in each field of ⫻330 magnification) in the urine, and all of them had telomerase activity using fluorescence-based TRAP assay (“positive/frequent” in Table 1 and Fig. 2A). It is noteworthy that 2 patients (Patients 4 and 7) had Class II/IIIa cytology, but both fluorescent-based TRAP and in situ TRAP assay demonstrated telomerase activity in the urine (Table 1). In these patients, bright fluorescent signals were detected in the nuclear portion. In Patient 9 (bladder carcinoma during irradiation), although TRAP failed to detect telomerase activity, cells with fluorescence signals only in the cytoplasmic area were detected (Fig. 2B). Cytoplasmic fluorescence without bright fluorescence in the nucleus is detected in some normal resting lymphocytes as well;19 thus, the signal may represent nonspecific fluorescence commonly observed in normal lymphocytes. In Patient 15 (prostate carcinoma during irradiation), although the TRAP assay failed to detect periodic 6 bp telomerase signals, a minor population of urinary cells had fluorescence in the nuclear area (“positive/ rare” in Table 1 and Fig. 2C). The dissociation similar to that observed in Patient 15 was also noted in 1 patient with bladder carcinoma at relapse (Patient 8), 2 with prostate carcinoma who underwent irradiation (Patients 15 and 16), and 1 with renal pelvic tumor before treatment (Patient 20). In patients with renal cell carcinoma, in-cell telomerase activity was detected only in a patient in whom telomerase activity was revealed by a fluorescence-based TRAP assay (Table 1). In 1 patient with ureter carcinoma (Patient 22) and 1 with renal stone (Patient 25), TRAP assay failed to detect telomerase activity; however, an in situ TRAP assay demonstrated granular fluorescent signals in the cytoplasmic area without bright fluorescence in the nuclear portion (Fig. 2D): the granular fluorescence positive cells without bright nuclear fluorescence might have been neutrophils because they had lobulated nuclei.19 Thus, this fluorescence pattern represented nonspecific staining for neutrophils without telomerase activity. We also analyzed 12 samples obtained from normal subjects and subjects with nonmalignant urologic disorders. Only 1 patient with renal stone had granular fluorescence in the cytoplasmic area without bright In-Cell Telomerase Activity in Urothelial Neoplasia/Ohyashiki et al. 2557 TABLE 1 Clinical Findings and Telomerase Activity in Exfoliated Urinary Cells from Patients with Urologic Disorders Telomerase activityb Diagnosis No. Bladder carcinoma 1 2 3 4 5 6 7 8 9 10 11 12 Prostate carcinoma 13 14 15 16 Renal cell carcinoma 17 18 Renal pelvic tumor 19 20 21 Ureter carcinoma 22 Lung carcinoma metastasis 23 Nonmalignant urologic disorders Renal stone 24 Hydronephrosis 25 Clinical stage Pathology (6 g) (0.6 g) In situ TRAPc No. of positive cells/no. of cells examined Age/gender Status Cytology (class)a 58/M 56/M 74/M 71/F 65/M 49/M 65/F 75/M 75/M 66/F 56/F 84/M Preoperation Preoperation Preoperation Preoperation Preoperation Prechemotherapy Prechemotherapy Relapse During irradiation During chemotherapy Postoperation Postoperation V V V II V V IIIa II II V II V T2N0M0 T3N0M0 T3bN0M0 T3bN0M0 T3N0M0 T3N1M0 T3N0M0 T3N0M0 T1N0M0 T3N1M0 T2N0M0 T1N0M0 TCC, G3⬎G2 TCC, G3 TCC, G3⬎G2 TCC, G2 TCC, G1⬎G2 TCC, G3 TCC, G2 TCC, G3 TCC, G3 TCC, G2 TCC, G3 TCC, G3 5.87 ND 0 ND ND 2.34 0 0 ND 1.35 ND ND 1.5 2.27 1.08 0.86 25.6 0.68 1.48 0 0 13.2 3.5 0 Positive/frequent Positive/frequent Positive/frequent Positive/rare Positive/frequent Positive/frequent Positive/frequent Positive/rare Cytoplasmic Positive/rare Positive/rare Negative 56/200 73/200 35/200 8/200 113/200 68/100 21/100 3/100 0/200 7/100 11/100 0/200 59/M Postoperation NA T2N0M0 0 0 Negative 0/200 72/M 82/M 69/M Chemotherapy During irradiation During irradiation II NA II T2N0M1 T3N0M0 T2N0M0 Well ⬍⬍ poorly diff. adeno Moderately diff. adeno Poorly diff. adeno Poorly diff. adeno ND ND ND 2.07 0 0 Positive/frequent Positive/rare Positive/rare 22/100 8/100 6/100 68/M 60/M Preoperation Preoperation II II T3N0M0V0 T3N0M0 Alveolar clear, G2 Alveolar clear, G2⬎G1 ND ND 0 30.03 Negative Positive/frequent 0/200 65/200 44/M 64/M 67/F Preoperation Preoperation Postoperative (Rel) IIIb IIIb II T2N0M0 T2N0Mx TaN0M0 TCC, G2⬎⬎G3 TCC, G2⬎G1⬎G3 TCC, G2 ND 0 ND 2.25 0 0 Positive/rare Positive/rare Negative 11/200 5/100 0/200 70/F Preoperation IIIb TCC, G3 0 0 Cytoplasmic granular 0/100 64/M Preoperation II Poorly diff. adeno ND 3.48 Positive/frequent 33/100 27/M IIIa ND 0 Cytoplasmic granular 0/100 65/M I ND 0 Negative 0/100 TRAP: telomeric repeat amplification protocol; TCC: transitional cell carcinoma; CHAP: cyclophosphamide, hexamethylmelamine, doxorubicin, and cisplatin; NA: no available data; ND: not done; diff. adeno: differentiated adenoma. a Cytology: The data at the time of examination of telomerase activity. b Telomerase activity: Parentheses indicate relative telomerase activity (the protein content in the CHAP extract). c In situ TRAP: Positive: positive cells for nuclear fluorescence; Cytoplasmic: cells with weak positive fluorescence in the cytoplasmic area without bright fluorescence at nucleus; Cytoplasmic granular: cells with granular positive fluorescence in the cytoplasmic area; Negative: only background fluorescence; Frequent: ⱖ3 cells with bright fluorescence in the field of 330 magnification; Rare: ⬍3 cells with bright fluorescence in the field of 330 magnification. nuclear fluorescence, and the remaining 11 had only weak background fluorescence. Association between Cytology and Telomerase Activity As shown in Table 1, 5 patients (Patients 1, 2, 3, 5, and 6) with bladder carcinoma and Class V cytology before treatment had telomerase activity in their exfoliated urothelial cells according to a fluorescence-based TRAP assay and a high frequency of telomerase positive cells according to an in situ TRAP assay. In contrast, some patients (Patients 4, 7, 8, 11, 14, 16, 18, 19, 20, and 23) with Class II/III cytology had in-cell telomerase activity 2558 CANCER December 15, 1998 / Volume 83 / Number 12 DISCUSSION FIGURE 1. Representative fluorocurves show telomerase activity (telomerase signals). Multiple peaks correspond to telomeric repeats that were synthesized by the presence of telomerase in exfoliated urothelial cells obtained from patients with urologic neoplasia and in a colon carcinoma cell line, COLO#320DM. To quantitate the telomerase activity, we used the internal standard showing its peak at 36 bp. The results analyzed by the Fragment Manager system and the area of telomerase signals is compared with the area of internal standard signal.21,22 In each subject, the size markers (50 bp and 100 bp) are shown on top. Lane 2: Patient 6 (bladder carcinoma, Class V cytology); Lanes 3 and 4: Patient 3 (bladder carcinoma, Class V); Lane 5: Patient 18 (renal cell carcinoma, Class II); Lane 6: Patient 14 (prostate carcinoma, Class II); Lane 7: Patient 23 (lung carcinoma metastasis, Class II); Lane 8: urine sample from a normal individual; Lane 9: COLO#320DM. In Patient 3, multiple peaks were detected after dilution (Lanes 3 and 4). In Patient 14 (prostate carcinoma, Class II cytology; Lane 6), small but detectable 6 bp periodic multiple peaks corresponding to telomerase activity were noted. in the urine, although for 3 of them (Patients 8, 16, and 20) fluorescence-based TRAP assay failed to detect telomerase activity. The dissociation might be due to the limitation of cytologic examination rather than contaminated lymphocytes, because all the patients with telomerase activity in their urine using a fluorescence-based TRAP assay had bright fluorescence positive cells with various frequencies using an in situ TRAP assay. We demonstrated that a fluorescence-based TRAP assay could detect telomerase activity semiquantatively in voided urine obtained from various urothelial neoplasia. None of the normal individuals had telomerase activity detected, indicating that the telomerase activity detected in the urinary sample may be derived from exfoliated cancers of the patients. Although Műller et al. failed to detect telomerase activity in naturally voided urine using either original TRAP or fluorescence-based TRAP assay.17 Yoshida et al. reported that 62% of the bladder carcinomas had telomerase activity in the exfoliated urine,18 supporting our results that in urinary samples from patients with urologic neoplasias, especially bladder carcinoma, telomerase activity can be detected. Yoshida et al. used protease inhibitors to determine urinary telomerase,18 in contrast to the report of Muller et al.17 We used freshly obtained voided urine and eliminated red blood cells that interfered with PCR by using hypotonic/hypertonic solution; thus, this procedure could be used to determine telomerase activity in the urothelial exfoliated cells from patients with bladder carcinoma. Our experiments using two different concentrations of protein in the CHAPS extract demonstrated the presence of PCR inhibitors in some samples, although Lin et al. did not find any bladder carcinoma that had PCR inhibitors.16 More recently, Kinoshita et al. reported that telomerase activity was detected in exfoliated cells in 23 of 42 voided urine specimens (55%) and in 36 of 43 bladder-washing fluid samples obtained from patients with bladder carcinoma, and they concluded that telomerase activity was more sensitive in detecting the presence of cancer than standard urine cytologic examination.24 We then applied an in situ TRAP assay to detect telomerase positive cells. We demonstrated that exfoliated urothelial cells from patients with various forms of urologic neoplasia showing telomerase activity using a fluorescence-based TRAP assay usually contained a high frequency of cells with bright fluorescence in the nuclei that may have corresponded to telomerase activity. In some patients without detectable telomerase activity using a TRAP assay, an in situ TRAP assay demonstrated a minor population of telomerase positive cells. Thus, an in situ TRAP assay to detect telomerase positive cells might be a powerful approach in the early detection and monitoring of urologic neoplasia during and after treatment. A high incidence of telomerase activity has been demonstrated in prostate carcinoma cases, 28 of 31 (90%) by Lin et al.25 and 21 of 25 (84%) by Sommerfeld et al.26 Although the number of urinary specimens In-Cell Telomerase Activity in Urothelial Neoplasia/Ohyashiki et al. 2559 FIGURE 2. The fluorescent signals shown correspond to telomerase activity in exfoliated urothelial cells obtained from patients with urologic neoplasia and from a normal individual. Many urinary cells (“positive/frequent” in Table 1) obtained from a patient with untreated bladder carcinoma (Patient 6) had bright fluorescence corresponding to telomerase activity (A). In Patient 12 (bladder carcinoma postoperation), only cells with cytoplasmic fluorescence without nuclear bright fluorescence were detected (B); possibly these cells were lymphocytes. In the urinary sample obtained from a patient with bladder carcinoma during chemotherapy (Patient 10), a minor population (“positive/rare” in Table 1) of urothelial cells showed bright fluorescent signals at the nuclei (C). Punctate fluorescence in cytoplasm without bright fluorescence in the nucleus was noted in the urinary specimen obtained from a patient with renal stone (Patient 25) (D). 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