Vol. 4, 1603-1608, July 1998 Clinical Cancer Research 1603 Advances in Brief Expression of Human Telomerase Subunits and Correlation with Telomerase Activity in Urothelial Cancer Hideaki Ito, Satoru Kyo,’ Taro Kanaya, Introduction Masahiro Takakura, Masaki Inoue, and Telomeres are the distal ends of eukaryotic chromosomes which in humans contain TTAGGG repeats (1). They protect Mikio Namiki and stabilize the ends of chromosomes (2). Because cellular Departments of Urology [H. I., M. N.] and Obstetrics and Gynecology DNA polymerases cannot replicate the 5’ end of linear DNA [S. K., T. K., M. T., M. I.], School of Medicine, Kanazawa University, Kanazawa, Ishikawa 920, Japan molecules, the number of tebomere repeats in normal somatic cells decreases with cell division (3). The shortening of te- lomeres results in chromosomal instability, which leads to cel- Abstract lular senescence. Telomerase is a specialized ribonucleoprotein The activation of telomerase and stabffization of te- polymerase containing an integral RNA with a short template bomeres are thought to be required for cellular immortality element that directs the de novo synthesis of telomeric repeats at and oncogenesis. Three major components of human tebom- chromosome ends (4, 5). Studies that use the PCR-based TRAP2 assay (6-9) have reported that telomerase is activated in a erase-human tebomerase RNA (hTERC), tebomerase-asso- variety of malignant tumors. In contrast, telomerase activity is ciated protein (TEP1), and human tebomerase catalytic sub- usually repressed in normal somatic tissues except in some unit (hTERT)-have recently been identified. However, the self-renewing tissues with high regenerative potential, such as roles played by these subunits in the regulation of tebomer- hematopoietic and endometrial cells (10, 1 1 ). Telomerase reac- ase activity are still unclear. In the present study, a total of tivation is thus thought to be required for stabilization of te- 37 urothelial cancers, including one metastatic lesion, and lomere length in attaining cellular immortality (12, 13). adjacent normal tissues as well as cell lines derived from Little is known, however, concerning the molecular mech- bladder cancers were examined for the expression of each anisms by which human telomerase is activated in tumors. This telomerase subunit. Reverse transcnption-PCR analysis re- is due mainly to the lack of findings concerning constituents of vealed that more than 90% of urothelial cancers expressed the human telomerase complex. hTERC was identified first and hTERT mRNA, whereas less than 20% of normal adjacent functions as a template for telomere elongation by telomerase tissues did. In contrast, hTERC and TEP1 mRNA were (14). Disrupting the function of telomerase RNA in Tetrahy- commonly expressed in both cancers and normal tissues. All mena through overexpression of an inactive form of telomerase of the three cell lines derived from bladder cancer expressed RNA has been shown to lead to progressive shortening of each of the tebomerase subunits, whereas the two normal telomeres (5). The introduction of antisense hTERC into tumor primary fibroblast cell lines expressed hTERC and TEP1 cells results in the loss of telomere sequences with subsequent mRNA but not hTERT mRNA. Telomerase activity was cellular senescence (14). These findings suggest that hTERC examined using tebomeric repeat amplification protocol as- plays an essential role in the maintenance of tebomeres. The say. All of the cancers examined exhibited tebomerase activ- level of hTERC expression has also been shown to increase with ity, whereas only 2 of 12 normal tissues exhibited weak tumor progression (15). Three proteins in different species as- activity. There was a significant association of tebomerase sociated with telomerase activity have also been identified. p80 activity with hTERT mRNA expression but not with hTERC and p95 were purified from ciliate Tetrahymena (16), and the or TEP1 mRNA expression. These findings provide strong gene encoding a mammalian homologue of p80, TEP1, has also evidence that the expression of hTERT is a rate-limiting been cloned (17, 18). However, recent studies have demon- strated that the expression of these telomerase-associated pro- determinant of the enzymatic activity of human tebomerase teins does not reflect the level of telomerase activity (19). The and that the up-regulation of hTERT expression may play a role of these proteins in the regulation of telomerase activity is critical role in human carcinogenesis. still unclear. Two related proteins, Est2p and p123, were iden- tified as catalytic subunits of telomerase in the yeast Saccharo- myces cerevisiae and the ciliate Euplotes aediculatus, respec- tively. Both proteins contain characteristic sequences in their genes that are well conserved in catalytic regions of reverse Received 1/22/98; revised 5/4/98; accepted 5/6/98. transcriptases (20, 21). The human homobogue of Est2p and The costs of publication of this article were defrayed in part by the p123 has most recently been cloned (hTERT) (22, 23). The payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I To whom requests for reprints should be addressed, at Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, 2 The abbreviations used are: TRAP, telomeric repeat amplification 13-1, Takaramachi, Kanazawa, Ishikawa 920, Japan. Phone: 8 1-0-76- protocol; hTERC, human telomerase RNA component; TEP1 , telomer- 265-2425; Fax: 8 1-0-76-234-4266; E-mail: [email protected] ase-associated protein; hTERT, human telomerase reverse transcriptase; u.ac.jp. RT-PCR, reverse transcription-PCR. Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 1998 American Association for Cancer Research. 1604 Expression of Telomerase Subunits in Urothelial Cancers expression of hTERT is observed at high levels in telomerase- by the Bradford assay (24). Five p.g of protein were used for the positive cell lines but not in telomerase-negative cell lines, TRAP assay. Assay tubes were prepared by sequestering 0.2 p.g which suggests that hTERT is a catalytic subunit homologue of CX primer (5’-CCCTFACCCTFACCCTFACCCTfAA-3’) protein. However, the expression of hTERT and its correlation under wax barrier (Ampliwax, Perkin-Elmer Corp., Foster City, with telomerase activity have not been determined for a large CA). Each extract was assayed in 50 p.1 of reaction mixture number of clinical samples. containing 20 mri Tris-HC1 (pH 8.0), 1 .5 nmi MgCl2, 60 mi In the present study, the expression of each telomerase KC1, 0.005% Tween 20, 1 mii EGTA, 50 p.M dNTPs, 0.2 i.g of subunit was examined in urothelial cancers and cell lines as well TS primer (5’-AATCCGTCGAGCAGAG1T-3’), 1 ig of T4g as in normal urothelial tissues, and the correlation between 32 protein (Boeringer-Mannheim), and 2.5 units of Taq DNA subunit expressions and telomerase activity was determined. We polymerase (Wako, Japan). After a 30-mm incubation at 23#{176}C found that hTERT was expressed in most of the cancers exam- for the telomerase-mediated extension of the TS primer, the med but not in normal urothelial tissues, whereas hTERC and reaction mixture was heated at 90#{176}Cfor 3 mm and then sub- TEP1 were broadly expressed both in cancers and in normal jected to 31 cycles of PCR including denaturation at 94#{176}Cfor tissues. A strong correlation was found between telomerase 45 s, annealing at 50#{176}Cfor45 s, and extension at 72#{176}Cfor 60 s. activity and the expression of hTERT, providing evidence that The PCR products were electrophoresed on a 12% polyacryl- hTERT functions as a critical determinant of the enzymatic amide gel and visualized with SYBR Green I nucleic acid gel activity of human telomerase. stain (FMC BioProducts, Rockland, ME). RNA-PCR Analysis. Total RNA was isolated from the tissues using Isogen (Nippon Gene, Tokyo, Japan) in accord- Materials and Methods ance with the manufacturer’s protocol. cDNA was synthesized Tissue Samples. Thirty-seven specimens of urothelial from 4 g of RNA using an RNA PCR Kit Version 2 (TaKaRa, tumors, including 20 bladder cancers, 1 metastatic lymph node, Tokyo, Japan) with random primers. Analysis of the expression 10 ureteral tumors, 5 renal pelvic tumors, and 1 renal cancer of each telomerase subunit was performed by RT-PCR ampli- with renal pelvic invasion, as well as 22 specimens of adjacent fication as described previously (22) with slight modification. normal tissue, were obtained at the time of surgery. The histo- To amplify the cDNA, l-i.l aliqots ofreverse-transcribed cDNA logical diagnoses were determined using sections of the same were subjected to PCR in 10 p.1 of 1 X buffer containing 0.2 mrvt specimens as used for RT-PCR. At the same institutions where each of dATP, dCTP, dGTP, and d1TP, and 0.6 units of ExTaq the surgeries and the diagnoses took place, detailed clinicopath- DNA polymerase (TaKaRa). A 0.45-i.l portion of DMSO (SIG- ological findings were evaluated according to the General Rules MA) was added to the buffer for hTERT. hTERT mRNA was for Clinical and Pathological Studies on Bladder Cancer, Renal amplified using the primer pair 5’-CGGAAGAGTGTCTG- Pelvic, and Ureteral Cancer, with the use of the tumor-node- GAGCAA-3’ (LT5) and 5’-GGATGAAGCGGAGTCTGGA-3’ metastasis classification system for malignant tumors. All of the (LT6); TEP1 mRNA was amplified using the primer pair 5’- samples were obtained within 2 h after surgical removal and TCAAGCCAAACCTGAATCTGAG-3’ (TEP1 . 1) and 5’CCC- were then frozen and stored at - 80#{176}Cuntil used for RT-PCR CGAGTGAATC1TFCTACGC-3’ (TEP1.2); and hTERC was and TRAP assay. amplified using the primer pair 5’-TCTAACCCTAACTGA- Urinary sediment tissue samples were also obtained from GAAGGGCGTAG-3’ (F3b) and 5’-GTVFGCTCTAGAAT- patients with and without bladder cancer from 50- and 150-nil GAACGQTGGAAG-3’ (R3c).