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0022-5347/99/1626-1889/0 THE JOURNAL OF UROLOGY® Copyright © 1999 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 162, 1889, December 1999 Printed in U.S.A.
This Month in Investigative Urology EXPRESSION OF THE CATALYTIC SUBUNIT ASSOCIATED WITH TELOMERASE GENE IN HUMAN URINARY BLADDER CANCER In 1973, Olovinikov proposed that cells lose a small amount of DNA at their terminal ends with each replication. The 39 to 59 (leading) strand of the parent DNA is copied in a continuous manner, but the 59 to 39 (lagging) strand of the parent DNA is copied discontinuously as Okazaki fragments. Each fragment is primed with an RNA primer, which is subsequently degraded. The fragments are then ligated by DNA repair enzymes that operate behind the replication fork. The 39 end of the lagging strand is then left incompletely copied and is lost. This piece of nucleotide is called a telomere. The cell can only afford to lose a finite number of these telomeres before significant sequences of the parent DNA are lost. Chromosomes lacking telomeres undergo fusions, translocations, and other rearrangements, leading to degradation by cellular enzymes. However, many cancer cells maintain their telomeres despite multiple rounds of replication. The telomere sequence is synthesized by a ribonucleoprotein called telomerase. In formal terms, telomerase belongs to the enzyme class of reverse transcriptase (RNA-directed DNA polymerases). Telomerase stabilizes telomere length by adding hexameric repeats, (TTAGGG)n, to the ends of the chromosome. The presence of telomerase activity may indicate the ability of a cell to bypass the telomeric “clock” that limits the proliferative capacity of normal somatic cells, and thus reflect the presence of immortal or cancer cells. The first investigators to demonstrate telomerase activity in vivo using tumor cells and non-malignant cells from end-stage ovarian carcinoma took advantage of telomerase’s ability to extend a (TTAGGG)n primer. This assay depended on direct detection of the products on telomerase action; when telomerase extends an exogenously supplied (TTAGGG)n primer, the products form a characteristic hexanucleotide ladder on gel electrophoresis. At least 107 to 108 cells were required for this assay, and the recovery of telomerase activity from extracts varied with cell type. Later a similar but more sensitive procedure involving the polymerase chain reaction and the telomeric repeat amplification protocol (TRAP) was developed. In the TRAP assay, telomerase synthesizes extension products, which then serve as the templates for PCR amplification. TRAP can detect telomerase activity in an amount of extract equivalent to 100 cells from a human immortal cell line. The TRAP assay has been applied to various urologic cancers, including human prostate and bladder cancer. Early reports detected telomerase activity in solid tissue specimens from bladder cancer at rates of 86% and 98%, regardless of tumor stage or differentiation, while telomerase activity was not detectable in normal bladder tissue specimens. The telomerase complex is composed of several subunits. The RNA subunit of telomerase, hTR, functions as a template for telomere elongation. This template in the RNA subunit codes for the hexameric repeats (TTAGGG). The subunit TP1 has been identified as a human homologue of Tetrahymena p80 and has been shown to associate with telomerase in vivo. The function of TP1 is yet to be determined, but it may be a regulatory component for telomerase activity. The human homologue of Est2protein, hEST2/hTRT, has recently been cloned. High levels of hTRT are observed in tumors and telomerase-positive cell lines but not in telomerase-negative cell lines, suggesting that hTRT may be a putative catalytic subunit homologue protein. Furthermore, disruption of the hTRT subunit, in in vitro telomerase reconstitution experiments, resulted in a nonfunctional telomerase enzyme. This month in Investigative Urology, Suzuki and associates examined the expression of two major components of the telomerase gene, hTRT and TP1. RT-PCR was performed on 27 human bladder cancers and 23 normal bladder tissues. hTRT expression was detected in all 27 (100%) cases, and TP1 was detected in 25 of 27 (93%) cases. In addition, 23 cases of normal bladder tissues showed no expression of hTRT, but 18 of 23 (78%) of normal bladder tissues showed TP1 expression. This finding strongly suggests that hTRT may be a useful subunit for the screening and diagnosis of urinary bladder cancer. The use of RT-PCR for the various subunits of telomerase may be a powerful method for noninvasive detection of bladder cancer. Early reports with the TRAP assay for noninvasive detection resulted in varied specificity and sensitivity. Telomerase activity has been detected in exfoliated cells in spontaneously voided urine specimens and in bladder-washing fluids with varying degrees of sensitivity of detection, using the TRAP assay in bladder cancer patients. We have also used the TRAP assay to determine the presence of telomerase activity in voided urine samples, and detected telomerase activity in 85% of bladder cancer specimens, while others reported sensitivity ranging around 70%. There are several reasons why a voided urine sample may not manifest telomerase activity as measured by TRAP. First, as stated in this month’s article by Suzuki and associates, the presence of inhibitors to the Taq polymerase and the polymerase chain reaction may cause false results. Furthermore, the samples that are not washed and processed within 24 hours of collection may undergo telomerase enzyme degradation. Perhaps the limitations of sample collection and/or processing may be overcome with the use of RT-PCR for these subunits. Understanding how these subunits are regulated and processed will certainly provide novel clinical applications. In the future, telomerase inhibition will likely be an innovative and exciting direction for chemoprevention or antitumor therapy. Agents that can inhibit telomerase activity and/or subunit processing may have clinical efficacy in intravesical therapy for bladder cancer. Brian C.-S. Liu, Ph.D. Molecular Urology Laboratory Brigham & Women’s Hospital Harvard Medical School Boston, Massachusetts 1889
Vol. 162, 1889, December 1999 Printed in U.S.A.
This Month in Investigative Urology EXPRESSION OF THE CATALYTIC SUBUNIT ASSOCIATED WITH TELOMERASE GENE IN HUMAN URINARY BLADDER CANCER In 1973, Olovinikov proposed that cells lose a small amount of DNA at their terminal ends with each replication. The 39 to 59 (leading) strand of the parent DNA is copied in a continuous manner, but the 59 to 39 (lagging) strand of the parent DNA is copied discontinuously as Okazaki fragments. Each fragment is primed with an RNA primer, which is subsequently degraded. The fragments are then ligated by DNA repair enzymes that operate behind the replication fork. The 39 end of the lagging strand is then left incompletely copied and is lost. This piece of nucleotide is called a telomere. The cell can only afford to lose a finite number of these telomeres before significant sequences of the parent DNA are lost. Chromosomes lacking telomeres undergo fusions, translocations, and other rearrangements, leading to degradation by cellular enzymes. However, many cancer cells maintain their telomeres despite multiple rounds of replication. The telomere sequence is synthesized by a ribonucleoprotein called telomerase. In formal terms, telomerase belongs to the enzyme class of reverse transcriptase (RNA-directed DNA polymerases). Telomerase stabilizes telomere length by adding hexameric repeats, (TTAGGG)n, to the ends of the chromosome. The presence of telomerase activity may indicate the ability of a cell to bypass the telomeric “clock” that limits the proliferative capacity of normal somatic cells, and thus reflect the presence of immortal or cancer cells. The first investigators to demonstrate telomerase activity in vivo using tumor cells and non-malignant cells from end-stage ovarian carcinoma took advantage of telomerase’s ability to extend a (TTAGGG)n primer. This assay depended on direct detection of the products on telomerase action; when telomerase extends an exogenously supplied (TTAGGG)n primer, the products form a characteristic hexanucleotide ladder on gel electrophoresis. At least 107 to 108 cells were required for this assay, and the recovery of telomerase activity from extracts varied with cell type. Later a similar but more sensitive procedure involving the polymerase chain reaction and the telomeric repeat amplification protocol (TRAP) was developed. In the TRAP assay, telomerase synthesizes extension products, which then serve as the templates for PCR amplification. TRAP can detect telomerase activity in an amount of extract equivalent to 100 cells from a human immortal cell line. The TRAP assay has been applied to various urologic cancers, including human prostate and bladder cancer. Early reports detected telomerase activity in solid tissue specimens from bladder cancer at rates of 86% and 98%, regardless of tumor stage or differentiation, while telomerase activity was not detectable in normal bladder tissue specimens. The telomerase complex is composed of several subunits. The RNA subunit of telomerase, hTR, functions as a template for telomere elongation. This template in the RNA subunit codes for the hexameric repeats (TTAGGG). The subunit TP1 has been identified as a human homologue of Tetrahymena p80 and has been shown to associate with telomerase in vivo. The function of TP1 is yet to be determined, but it may be a regulatory component for telomerase activity. The human homologue of Est2protein, hEST2/hTRT, has recently been cloned. High levels of hTRT are observed in tumors and telomerase-positive cell lines but not in telomerase-negative cell lines, suggesting that hTRT may be a putative catalytic subunit homologue protein. Furthermore, disruption of the hTRT subunit, in in vitro telomerase reconstitution experiments, resulted in a nonfunctional telomerase enzyme. This month in Investigative Urology, Suzuki and associates examined the expression of two major components of the telomerase gene, hTRT and TP1. RT-PCR was performed on 27 human bladder cancers and 23 normal bladder tissues. hTRT expression was detected in all 27 (100%) cases, and TP1 was detected in 25 of 27 (93%) cases. In addition, 23 cases of normal bladder tissues showed no expression of hTRT, but 18 of 23 (78%) of normal bladder tissues showed TP1 expression. This finding strongly suggests that hTRT may be a useful subunit for the screening and diagnosis of urinary bladder cancer. The use of RT-PCR for the various subunits of telomerase may be a powerful method for noninvasive detection of bladder cancer. Early reports with the TRAP assay for noninvasive detection resulted in varied specificity and sensitivity. Telomerase activity has been detected in exfoliated cells in spontaneously voided urine specimens and in bladder-washing fluids with varying degrees of sensitivity of detection, using the TRAP assay in bladder cancer patients. We have also used the TRAP assay to determine the presence of telomerase activity in voided urine samples, and detected telomerase activity in 85% of bladder cancer specimens, while others reported sensitivity ranging around 70%. There are several reasons why a voided urine sample may not manifest telomerase activity as measured by TRAP. First, as stated in this month’s article by Suzuki and associates, the presence of inhibitors to the Taq polymerase and the polymerase chain reaction may cause false results. Furthermore, the samples that are not washed and processed within 24 hours of collection may undergo telomerase enzyme degradation. Perhaps the limitations of sample collection and/or processing may be overcome with the use of RT-PCR for these subunits. Understanding how these subunits are regulated and processed will certainly provide novel clinical applications. In the future, telomerase inhibition will likely be an innovative and exciting direction for chemoprevention or antitumor therapy. Agents that can inhibit telomerase activity and/or subunit processing may have clinical efficacy in intravesical therapy for bladder cancer. Brian C.-S. Liu, Ph.D. Molecular Urology Laboratory Brigham & Women’s Hospital Harvard Medical School Boston, Massachusetts 1889