760 Telomerase Activity in Renal Cell Carcinoma Ken-ichiro Yoshida, M.D. Shu-ichi Sakamoto, M.D. Shuhei Sumi, M.D. Yotsuo Higashi, M.D. Satoshi Kitahara, M.D. Department of Urology, Dokkyo University School of Medicine, Tochigi, Japan. BACKGROUND. Telomerase activity has been shown to be increased in numerous tumors and cell lines, although to the authors’ knowledge there has been no previous assessment of telomerase activity in renal cell carcinoma (RCC). To examine whether telomerase activity could be used as a biochemical parameter for predicting the behavior of RCC, telomerase activity was quantified in RCC samples and correlated with clinicopathologic findings. METHODS. Telomerase activity was quantified in 50 RCC samples and 21 samples of normal renal tissue using fluorescence-based telomeric repeat amplification. RESULTS. Telomerase activity was detected in 28 of 50 RCC samples (mean 6 standard deviation, 35.03 6 18.47 U/mg protein), whereas there was no telomerase activity in 21 normal renal tissue specimens. Telomerase activity in tumors classified as pT4 was significantly higher than in tumors classified as pT1 to pT3. Telomerase activity also correlated with patient age. CONCLUSIONS. Telomerase activity was increased in RCC samples compared with normal renal tissues. Although significant associations were observed between telomerase activity and clinicopathologic parameters, further study is required to determine whether telomerase activity affects the biologic and clinical behavior of RCC. Cancer 1998;83:760 – 6. © 1998 American Cancer Society. KEYWORDS: telomerase activity, renal cell carcinoma, TNM classification, clinicopathologic parameters. R Address for reprints: Ken-ichiro Yoshida, M.D., Department of Urology, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu, Shimotsuga-gun, Tochigi 321-02, Japan. Received September 29, 1997; revision received January 12, 1998; accepted February 2, 1998. © 1998 American Cancer Society enal cell carcinoma (RCC) is a tumor that varies in its biologic and clinical behavior.1–3 The biologic behavior of RCC is difficult to predict from histologic findings.4 Because recurrence and metastasis can occur years after the primary presentation of RCC and there can be major differences in clinical outcome even within the same tumor stages, additional prognostic indicators are needed. Several previous studies have focused on genes believed to be involved in autocrine and paracrine mechanisms of tumor proliferation and progression.5–9 In this study the activity of the enzyme telomerase was assessed as a means of better defining the behavior of RCC. This enzyme is a ribonucleoprotein that elongates telomeric sequences.10 These sequences are specialized structures of characteristic repetitive DNA sequences that form specialized nucleoprotein complexes at the ends of eukaryotic chromosomes. Possible functions of telomere sequences include prevention of chromosome degradation, end-to-end fusions, rearrangements, and chromosome loss.11 Human telomeres undergo progressive shortening with each cell division through replication-dependent sequence loss at DNA termini.12 It is believed that dividing cells undergo only a limited number of replication cycles unless they activate the telomerase enzyme.13 Recent studies have demonstrated that a variety of cell lines and malignant tumors have telomerase activity, whereas normal somatic cells do not. This suggests that telomerase activation is a critical step in cell immortaliza- Renal Cell Carcinoma and Telomerase Activity/Yoshida et al. tion and oncogenesis.14 –16 Recent studies also have demonstrated that telomerase activity can be a useful indicator of clinical outcome.15,17–21 In this study telomerase activity in RCC was quantified using fluorescence-based telomeric repeat amplification (TRAP) and the TRAP-eze telomerase detection kit (Oncor, Inc., Gaithersburg, MD).22–25 The relation between telomerase activity and clinicopathologic findings then was examined. MATERIALS AND METHODS Patients and Tumors Fifty previously untreated patients with primary RCC (35 men and 15 women) diagnosed from January 1993 through April 1997 were studied. The median age was 55 years (range, 31–75 years). All patients underwent radical nephrectomy with lymph node dissection. Fifty tumor samples, free from necrotic tissues, and 21 normal tissue samples were obtained from the RCC patients. Normal renal tissues were defined histologically. All patients consented to the use of their renal tissues for the current study. After removal of the tumor, a specimen was frozen immediately in the operating room and stored at 280 °C until use. TNM staging of the tumors was determined according to the International Union Against Cancer (UICC) and the American Joint Committee on Cancer (AJCC) staging systems.26 Microscopic sections were evaluated by a pathologist without knowledge of patient clinical characteristics. The classification of the tumor was determined according to the UICC/AJCC.27 Nuclear grade was determined according to Fuhrman et al. using the worst area identified.28,29 Of 50 patients, 13 (26%) had distant metastasis. Distant metastasis were present in the lung alone in seven patients, the lung and liver in three patients, and the lung and other sites in three patients. Eight patients died of disease with a median survival of 8 months; 42 were alive at last follow-up. Patient follow-up ranged from 3– 46 months (median follow-up, 17 months). Determination of Telomerase Activity Telomerase activity was quantified with the TRAPeze telomerase detection kit (Oncor Inc.) according to the manufacturer’s instructions. In brief, frozen samples (50 mg) were homogenized in 200 mL of icecold 3-[(3-cholamidopropyl)dimethylammonio)]-1propanesulfonate (CHAPS) lysis buffer (containing 10 mM Tris-HCl [pH 7.5], 1.5 mM MgCl2, 1 mM ethyleneglycoltetraacetic acid [EGTA], 0.1 mM benzaminide, 5 mM 2-mercaptoethanol, 0.5% CHAPS, and 10% glycerol) and were incubated for 30 minutes on ice. After incubation the lysates were centrifuged at 12,000 3 g for 20 minutes at 4 °C. The 761 resulting supernatant fluids were used in the telomerase assay. The protein concentration was determined with the Coomassie Protein Assay Reagent (Bio-Rad, Hercules, CA), and an extract aliquot containing 1 mg of protein was used for each telomerase assay. Extracts were incubated with 0.1 ng fluorescence-labeled TS forward primer (59-AATCCGTCGAGCAGAGTT-39; Pharmacia Biotech, Uppsala, Sweden) in a master mix; the reaction buffer contained 20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCL, 0.005% Tween-20, 1 mM EGTA, 50 mM deoxynucleoside triphosphates, 1 mg T4 gene 32 protein (Boehringer-Mannheim Biochemicals, Indianapolis, IN), 0.01% bovine serum albumin, 2 U of Taq DNA polymerase (Takara Shuzo, Kyoto, Japan), and 0.5–5 mg of the CHAPS cell extracts. After a 30-minute incubation at 30 °C, 30 cycles of polymerase chain reaction (PCR) were performed (94 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 45 seconds). The products were diluted with an equal volume of formamide dye solution, heated at 94 °C for 5 minutes, and 5 mL was applied to each lane of a 10% denaturing gel containing 6 M urea. The gel was fitted to an automated DNA sequencer (ALF Red DNA Sequencer II; Pharmacia Biotech). The temperature of the gel was maintained at 45 °C during electrophoresis at 45 watts. The data from the DNA sequencer were collected and analyzed automatically using Fragment Manager VI. I software (Pharmacia Biotech). Each peak was quantified in terms of size, peak height, and peak area. Telomerase positive cell extracts provided in the kit (TSR 8) were used as a positive control. Telomerase activity was determined as telomerase values relative to those of a positive control: (measured total area of telomerase activity [50 base pair (bp), 56 bp, 62 bp, 68 bp, etc.])/(measured area of internal control [36 bp]) 3 (measured total area of telomerase activity [50 bp, 56 bp, 62 bp, 68 bp, etc.] in positive control)/(measured area of internal control [36 bp] in positive control) 3 100. Telomerase activity was expressed as total product generated (TPG) units/mg protein. All samples were assayed in duplicate and the mean value was determined. Statistical Analysis Values were expressed as the mean 6 standard deviation. Statistical analyses were performed using the Mann–Whitney U test and chi-square test. Correlations were examined by Spearman’s rank correlation procedure. P , 0.05 was considered to be statistically significant. All statistical analyses were performed using Stat View data analysis software (Abacus Concepts Inc., Berkeley, CA). 762 CANCER August 15, 1998 / Volume 83 / Number 4 FIGURE 1. A representative assay of telomerase activity in renal cell carcinoma tissues using the TRAP (telomeric repeat amplification)-eze telomerase detection kit. (Oncor, Inc., Gaithersburg, MD) The first peak is the fluorescence-labeled TS primer. The second peak is the internal control (36 base pair [bp]). The polymerase chain reaction product of telomerase extension yielded a 6-nucleotide peak profile (50 bp, 56 bp, 62 bp, 68 bp, 74 bp, etc.). RESULTS Telomerase Activity and Clinicopathologic Correlations A representative telomerase activity assay using pooled RCC tissues is shown in Figure 1. The first peak is fluorescence-labeled TS. The second peak is the internal control (36 bp). The telomerase activity was detected from the third peak in a shark-tooth pattern, extending from 50 bp with a 6-nucleotide periodicity (50 bp, 56 bp, 62 bp, 68 bp, 74 bp, etc.). The calculated telomerase activity in this case was 44.31 U/mg protein. Table 1 summarizes the clinicopathologic and telomerase activity data of 50 RCC cases. Telomerase activity was detected in 28 of 50 RCC samples (56%). Telomerase activity in RCC specimens ranged from 0 –94.26 U/mg protein, with a mean value of 19.67 6 23.28 U/mg protein. Telomerase activity was not detected in 21 normal renal tissues. The telomerase activity of these paired samples are shown in Table 2. The histologic findings in 22 RCC cases without telomerase activity were as follows: Cases 3, 12, 3, 4, and none were staged as pT1, pT2, pT3a, pT3b, and pT4, respectively; Cases 12, 5, 5, and none were staged as pV0, pV1a, pV1b, and pV2, respectively; Cases 20 and 2 were staged as pN0 and pN1, respectively; and Cases 17 and 5 were staged as pM0 and pM1, respectively. Fifteen were of conventional type, 4 were of papillary type, and 3 were of chromophobic type. Eight were nuclear Grade 1, 11 were nuclear Grade 2, 3 were nuclear Grade 3, and none was nuclear Grade 4. The RCC samples without telomerase activity did not have any distinct differences in histopathologic findings compared with RCC samples with telomerase activity. Comparative studies of telomerase activity in various histopathologic states of the tumor are summa- rized in Table 3. There was no significant difference in the mean telomerase activity between male (21.30 6 22.03 U/mg protein; n 5 35) and female patients (15.69 6 23.13 U/mg protein; n 5 15). Telomerase activity was not detected in the pT1 samples (n 5 2). The mean telomerase activity in the pT2 samples was 17.55 6 20.82 U/mg protein (n 5 27). The mean telomerase activity in the pT3a samples was 24.02 6 18.38 U/mg protein (n 5 14), whereas the enzyme activity was not detected in the pT3b samples (n 5 3). The mean telomerase activity was highest in the pT4 samples (48.66 6 31.37 U/mg protein; n 5 4), and this was significantly higher than in the pT2 (P 5 0.0275) and pT3 samples (P 5 0.0285). In contrast, no significant differences were observed among pT1, pT2, and pT3 samples. The mean telomerase activity in pV0 tumors was 17.31 6 20.65 U/mg protein (n 5 25), and was 26.35 6 26.48 U/mg protein (n 5 16) and 14.04 6 17.46 U/mg protein (n 5 9) in pV1a and pV1b tumors, respectively. The mean value of telomerase activity in pN0, pN1, and pN2 tumors was 19.00 6 22.93 (n 5 45), 32.35 6 10.44 (n 5 3), and 14.39 6 20.34 U/mg protein (n 5 2), respectively. The mean value of telomerase activity in pM0 tumors was 20.07 6 24.32 U/mg protein (n 5 37), which was similar to the mean value of 18.33 6 15.84 U/mg protein (n 5 13) for pM1 tumors. The mean telomerase activity in conventional type tumors (21.18 6 23.18 U/mg protein; n 5 37) was highest, followed by papillary type (18.90 6 21.45 U/mg protein; n 5 9) and chromophobic type tumors (6.81 6 13.62 U/mg protein; n 5 4). The mean value of telomerase activity in nuclear Grade 1 tumors was 21.14 6 25.38 U/mg protein (n 5 19), and, in decreasing order, was 19.48 6 20.83 U/mg protein for nuclear Grade 2 tumors (n 5 26) and 14.53 6 20.70 U/mg protein for nuclear Grade 3 tumors (n 5 5). The mean telomerase activity in Stage I and II tumors had similar values (Stage I: 17.13 6 34.25 U/mg protein [n 5 4] and Stage II: 15.84 6 19.20 U/mg protein [n 5 22]), whereas the mean telomerase activity of Stage III and IV tumors was slightly higher than that of Stage I and II tumors (Stage III: 21.02 6 21.18 U/mg protein [n 5 11] and Stage IV: 25.58 6 25.43 U/mg protein [n 5 13]). No significant differences in telomerase activity were noted among these various histopathologic parameters (Table 3). Relations between telomerase activity and patient age, nuclear grade, or tumor stage also were studied. A significant correlation was observed between telomerase activity and patient age (P 5 0.0250) (Fig. 2), whereas no significant correlation was observed between telomerase activity and nuclear grade or tumor stage. At last follow-up eight patients had died of their Renal Cell Carcinoma and Telomerase Activity/Yoshida et al. 763 TABLE 1 Clinicopathologic Findings and Telomerase Activity in Renal Cell Carcinoma Case no. Gender Age (yrs) TNM staging Classification Nuclear grade Stage Telomerase activitya Clinical outcome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 M M F F M M M M M M M F F M M F M F M M F F F M M F M M M M M M M F M M F F M M M M M M F M M M M F 66 55 61 73 67 58 64 74 36 55 41 49 54 74 62 53 52 56 62 31 52 72 50 70 38 75 43 49 54 63 60 55 44 48 73 49 61 44 46 62 55 62 69 51 65 76 56 63 66 70 pT2pV1aN0M0 pT3bpV1bN0M1 pT3apV1aN0M0 pT2pV1aN0M0 pT2pV0N0M0 pT2pV0N1M1 pT2pV0N0M0 pT2pV0N0M0 pT3bpV1bN2M1 pT2pV0N0M0 pT2pV1aN0M0 pT3bpV1aN2M0 pT2pV0N0M0 pT3apV0N0M0 pT3apV1aN0M0 pT2pV0N0M0 pT2pV1aN0M0 pT1pV1aN0M0 pT2pV0N0M0 pT2pV0N0M0 pT3bpV1bN0M1 pT4pV1bN1M1 pT2pV0N0M0 pT3apV1aN0M1 pT2pV1aN0M0 pT3apV1aN0M0 pT3apV1aN0M1 pT3apV1aN0M0 pT3apV0N0M1 pT4pV1aN0M0 pT4pV1bN2M1 pT3apV0N0M0 pT3apV1bN0M1 pT2pV0N0M0 pT4pV1bN1M1 pT2pV0N0M0 pT1pV0N0M0 pT2pV1aN0M0 pT1pV0N0M0 pT2pV0N0M0 pT3apV1bN0M0 pT2pV1aN0M1 pT2pV0N0M0 pT3apV1aN0M0 pT2V0N0M0 pT2V0N0M0 pT2V0N0M0 pT2V1aN0M0 pT2V0N0M0 pT2V0N0M1 Conventional Conventional Conventional Conventional Conventional Conventional Conventional Papillary Papillary Conventional Conventional Conventional Chromophobic Chromophobic Papillary Papillary Conventional Conventional Papillary Conventional Chromophobic Chromophobic Conventional Papillary Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Papillary Conventional Conventional Conventional Conventional Conventional Papillary Papillary 1 2 2 2 1 2 1 2 2 1 2 3 1 2 2 2 2 2 2 3 2 2 3 3 1 2 1 1 2 1 2 1 2 1 3 1 2 1 1 1 1 2 2 1 1 2 2 2 1 2 II III III II II IV II II IV II III III II III III II II I II II IV IV II IV II III IV III IV IV IV III IV II IV II I II I II III IV II III I II II II I IV 18.52 0 20.21 44.31 56.42 25.45 20.65 26.86 0 0 52.46 0 0 0 61.64 0 0 0 0 0 0 27.23 0 28.31 0 20.74 29.06 21.72 23.68 94.26 28.77 28.40 0 54.28 44.36 0 68.50 0 0 34.40 26.03 31.46 35.40 0 0 11.83 0 28.01 17.90 0 Alive DOD Alive Alive Alive Alive Alive Alive DOD Alive DOD DOD Alive Alive Alive Alive Alive Alive Alive Alive DOD Alive Alive DOD Alive Alive Alive Alive Alive Alive DOD Alive Alive Alive DOD Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive M: male; DOD: died of disease; F: female. a Telomerase activity is expressed as U/mg protein. disease. Telomerase activity was detected in the tumors from four of these patients. The mean value of telomerase activity in those who died of RCC was 19.24 6 22.0 U/mg protein (n 5 8) compared with a mean value of 19.69 6 22.60 U/mg protein (n 5 42) among the patients still alive at last follow-up. No 764 CANCER August 15, 1998 / Volume 83 / Number 4 TABLE 2 Telomerase Activity in the Paired Samples of Normal and Carcinoma Tissues of the Same Kidney TABLE 3 Relation between Telomerase Activity and Clinicopathologic Features of Renal Cell Carcinoma Telomerase activity (U/mg protein) Case no. a 1 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 37 46 47 48 49 a Carcinoma Tissue Normal Tissue 18.52 0 20.21 56.42 25.45 20.65 26.86 0 0 52.46 0 0 0 61.64 0 0 68.50 11.83 0 28.01 17.90 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Case no. is the same as that in Table 1. significant difference in telomerase activity was observed between the two groups. Gender Male Female TNM staging pT1 pT2 pT3 pT4 pV0 pV1a pV1b N0 N1 N2 M0 M1 Stage I II III IV Nuclear grade 1 2 3 Tumor classification Conventional Papillary Chromophobic Telomerase activitya No. of cases Statistical analysis 21.30 6 22.03 15.69 6 23.13 35 15 NS 0 17.55 6 20.82 18.37 6 19.07 48.66 6 31.37 17.31 6 20.65 26.35 6 26.48 14.04 6 17.46 19.00 6 22.93 32.35 6 10.44 14.39 6 20.34 20.07 6 24.32 18.33 6 15.84 17.13 6 34.25 15.84 6 19.20 21.02 6 21.18 25.58 6 25.43 2 27 17 4 25 16 9 45 3 2 37 13 4 22 11 13 21.14 6 25.38 19.48 6 20.83 14.53 6 20.70 19 26 5 NS 21.18 6 23.18 18.90 6 21.45 6.81 6 13.62 37 9 4 NS pT2 vs. pT4: P 5 0.0275 pT3 vs. pT4: P 5 0.0285 NS NS NS NS NS: not significant. a Telomerase activity is expressed as U/mg protein. Assessment of Telomerase Negative Tumors It is possible that the absence of telomerase activity in a tumor may result from the presence of an inhibitor. We examined this hypothesis by mixing extracts from each of 22 telomerase negative tumors with the extract from positive control cells. In no cases did inhibition of the telomerase activity of positive control cells occur. DISCUSSION Telomeres are the specialized structures that contain TTAGGG repeats at the ends of all eukaryotic chromosomes. They are believed to contribute to chromosome stabilization by protecting genomic DNA against degradation and deleterious recombination events.10,30 Using a newly developed PCRbased telomerase assay, researchers have found that a variety of cell lines and malignant tumors express telomerase activity,14 –21 unlike normal somatic cells. This suggests that telomerase activation may be a critical step in cell immortalization and oncogenesis. The TRAP method generally has been used for the FIGURE 2. Relation between telomerase activity and patient age. A significant correlation was observed between telomerase activity and patient age in renal cell carcinoma (correlation coefficient 5 0.317; P 5 0.0250). detection of telomerase activity. However, it is a timeconsuming procedure that uses a radioisotope labeling system and has difficulties with regard to quantitation. To overcome these problems we used the Renal Cell Carcinoma and Telomerase Activity/Yoshida et al. TRAP-eze telomerase detection kit in the current study. In this method telomerase activity is represented by fluorescence curves, and the peak area is calculated automatically. The introduction of an internal standard allows telomerase activity to be measured in real time.21–25 The current study demonstrated telomerase activity in 28 of 50 RCC samples but no activity in normal renal tissues. These findings suggest that activation of telomerase may be a sign of immortalization and an important feature of RCC oncogenesis. Our results also show that telomerase is not always activated in RCC. Because we did not demonstrate telomerase activity inhibitors in telomerase negative RCC samples, it appears that telomerase activity is not the only factor regulating telomere length and stability. Murnane et al.31 and Bryan et al.32 have reported a telomeraseindependent mechanism in immortalized human cell lines that do not contain detectable telomerase activity. Recently, the experimental data from telomerase knock-out mouse suggest that telomerase is not required for oncogenic transformation or tumor formation.33 Some other organisms also are known to have telomerase-independent means of lengthening their telomeres. The yeast Saccharomyces cerevisiae has the ability to utilize recombination as a backup mechanism for telomere repair. Nonreciprocal recombination at the boundary between telomeric and nontelomeric DNA resulted in the acquisition of telomeric repeats by very short telomeres in this organism.34 The telomeres of Drosophila are maintained by transposition of specialized retrotransposons to broken or natural chromosome ends.35–37 None of these mechanisms as yet have been demonstrated in RCC. Recent studies have reported a positive correlation between telomerase activity and clinicopathologic findings for certain types of tumors.14,15,17,19,38 Lin et al.19 reported that telomerase activity was associated with pathologic grade and clinical stage in bladder carcinoma. Chadeneau et al.14 reported an association between telomerase activity and the acquisition of malignancy in colorectal carcinoma, and Hohaus et al.38 reported an association with disease stage. In the current study we observed an association between telomerase activity and pT. This suggests that telomerase activity in RCC may occur as a later event in cancer progression, similar to a report in nonsmall cell lung carcinoma.16 Moreover, recent studies have reported associations between telomerase activity and patient outcome in gastric carcinoma15 and neuroblastoma.20 In the current study we did not find an association between telomerase activity and patient outcome. However, the follow-up period in the current study was too short to draw definitive conclusions 765 regarding a possible relation between telomerase activity and patient outcome in RCC. In the current study we also demonstrated that RCC tumors in older patients have higher telomerase activity than those in younger patients. 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