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Clinical Chemistry 44:12 2446 –2452 (1998)

Enzymes and Protein Markers

New method to measure telomerase activity by transcription-mediated amplification and hybridization protection assay Minoru Hirose,1* Junko Abe-Hashimoto,1 Hidetoshi Tahara,2 Toshihide Ide,2 and Tadashi Yoshimura1

Telomerase is a ribonucleoprotein complex that uses RNA as a template for the addition of telomeric repeats. The development of the telomeric repeat amplification protocol (TRAP), a sensitive PCR-based assay, has facilitated the detection of telomerase activity in small tissue and tumor samples. Telomerase activity is expected to be a new diagnostic and prognostic marker of human cancer. In this study, we applied a non-PCR-based transcription-mediated amplification (TMA) and hybridization protection assay (HPA) to the measurement of telomerase activity by modification of both primers in TMA. We demonstrated that the modified TMA can detect and measure telomerase activity. TMA/HPA is as sensitive and reproducible as conventional TRAP, but is both faster and easier to perform. Furthermore, we found that TMA/HPA was influenced minimally by TRAP inhibitors that may come from clinical samples. TMA/HPA, which is easy, rapid, and applicable to a high-throughput format, should be clinically useful for the detection and monitoring of telomerase activity. Telomerase is a riboprotein that synthesizes and directs the addition of telomeric repeats onto the 39 end of existing telomeres, using its RNA component as template (1–3). Telomerase is thought to be important in the protection and replication of chromosomes (4), and its activity may play a role in events related to cellular immortality. In contrast to somatic cells, germ line cells are immortal and preserve the full genomic information for transfer to offspring organisms. Thus, telomerase

1 Diagnostics Science Laboratory, Chugai Diagnostics Science Co., Ltd., 3-41-8 Takada, Toshima-ku, Tokyo 171-8545, Japan. 2 Department of Cellular and Molecular Biology, Hiroshima University School of Medicine, 1-2-3 Kasumi, Hiroshima 734-0037, Japan. *Author for correspondence. Fax 81-3-3989-0785; e-mail hirosemnr@ chugai-pharm.co.jp. Received June 11, 1998; revision accepted September 10, 1998.

catalyzes the addition of TTAGGG repeats to the end of vertebrate chromosomes in germ cells, and telomerase activity is expressed in most immortalized cell lines and tumors (5–7). The conventional primer extension-based assay for detecting telomerase activity requires large amounts of sample and only allows detection of telomerase activity with limited sensitivity (2). The telomeric repeat amplification protocol (TRAP),3 which is a more sensitive PCR-based assay, overcomes those disadvantages (8). TRAP can detect telomerase activity in a small tissue sample or tumor biopsy (9 –12). However, conventional TRAP has some disadvantages, particularly for clinical use: it is time-consuming to analyze amplification products by polyacrylamide gel electrophoresis; and it is necessary to measure the area or intensity of 6-base ladders by densitometry with a computer program for quantitative analysis. Therefore, conventional TRAP can analyze only a limited number of samples. Another disadvantage is that the method is susceptible to inhibition from extracts of clinical samples. Because of the high frequency of this problem, internal controls are routinely added to TRAP assays to determine whether the PCR reactions are inhibited (13, 14). We previously reported that the hybridization protection assay (HPA) in conjunction with TRAP is a useful tool for detecting and measuring telomerase activity for mass diagnosis (15). HPA, which uses an acridinium ester-labeled probe, is a homogeneous assay and does not require radioactive material (16, 17). In addition, HPA is rapid, sensitive, and easy to quantify without computer programs, which dramatically simplifies the detection step. However, HPA alone cannot solve the problem of

3 Nonstandard abbreviations: TRAP, telomeric repeat amplification protocol; HPA, hybridization protection assay; TMA, transcription-mediated amplification; HCC, hepatocellular carcinoma; CHAPS; 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate; AE, acridinium ester; and rlu, relative light units.

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PCR-based TRAP inhibitors. Therefore, we tried to apply the non-PCR-based transcription-mediated amplification (TMA) protocol developed by Gen-Probe Inc., which is an isothermal system and can be performed in a heat block or water bath (18, 19). The kinetics of TMA are very rapid, and billions of RNA amplicons are produced from a single target molecule in ,1 h. TMA can be used with any type of nucleic acid target, including rRNA, mRNA, or DNA. We found that TMA was influenced minimally by the inhibitors under certain conditions. Here we report the establishment of a combined TMA/ HPA method to measure telomerase activity.

Materials and Methods cell lines and tissues K562 erythroid leukemia cells and HL60 promyelocytic leukemia cells were maintained in RPMI-1640 supplemented with 100 mL/L heat-inactivated fetal calf serum at 37 °C in a humidified atmosphere of 5% CO2 in air. Liver and colorectal samples were obtained from 81 and 10 patients, respectively, at Hiroshima University Hospital and affiliated hospitals as reported previously (20). Hepatocellular carcinomas (HCCs) were histologically classified as moderately or poorly differentiated HCCs. All colorectal samples, which have been shown to include inhibitors for conventional TRAP, were used to determine the effect of inhibitors on TMA/HPA. Extracts from cell lines and tissue samples were prepared by the 3 - [(3 cholamidopropyl) - dimethylammonio] - 1 - propane -sulfonate (CHAPS) detergent method as described previously (8).

tma assay The mechanism of TMA is described in Fig. 1A (18, 19). TMA uses two primers and two enzymes: T7 RNA polymerase and reverse transcriptase. In practice, as shown in Fig. 1B, the TMA/HPA procedure is very simple and fast. The CHAPS extract (2 mL) was mixed with 48 mL of TMA mixture I [40 mmol/L Tris-HCl, pH 7.5, 20 mmol/L MgCl2, 17.5 mmol/L KCl, 2 mmol/L each dNTP, 50 g/L polyvinylpyrrolidone, and 200 nmol/L promoter primer (59-AAT TTA ATA CGA CTC ACT ATA GGG AGA CTC TCT CTC TCT CTC TCT CTA GAG TT-39)] and incubated at 20 °C for 30 min, and then 25 mL of TMA mixture II [80 mmol/L Tris-HCl, pH 7.5, 32 mmol/L MgCl2, 14.8 mmol/L KCl, 16 mmol/L each rNTP, 100 g/L polyvinylpyrrolidone, and 1.2 mmol/L reverse primer (59-TTA CCC TTA CCC TTA CCC T-39)] was added. After the mixture was incubated at 94 °C for 5 min and cooled at room temperature for 5 min, 25 mL of the enzyme mixture, which contained Moloney murine leukemia virus (M-MuLV; United States Biochemical; 1000 units/assay) and T7 RNA polymerase (600 units/ assay; Epicentre Technologies), was added.The amplification reaction was performed at 40 °C for 75 min. To determine whether the TMA/HPA signals were dependent on telomerase activity, the mixture was treated

Fig. 1. Mechanism of TMA (A) and TMA/HPA procedure (B). (A) The mechanism of TMA is as follows: (step 1) The promoter primer consists of a promoter sequence for T7 RNA polymerase and a telomerase substrate sequence. Telomerase catalyzes the addition of TTAGGG repeats to the 39 end of the promoter primer. (step 2) Reverse primer hybridizes to a single-stranded DNA elongated by telomerase, and then reverse transcriptase (RTase) creates a double-stranded DNA. (step 3) T7 RNA polymerase recognizes the promoter sequence in the DNA template and initiates transcription. (step 4) Reverse primer hybridizes to synthesized RNA. Reverse transcriptase creates a DNA copy of the synthesized RNA by extension from the 39 end of the reverse primer. The RNA of the RNA:DNA duplex is degraded by the RNase H activity of reverse transcriptase. The promoter primer hybridizes to the DNA copy, and a doublestranded DNA is generated by reverse transcriptase (RTase) again. (step 5) Because each DNA template can make 100-1000 copies of the RNA amplicon, this expansion can produce 10 billion amplicons. The entire process is autocatalytic and is performed at one temperature. (step 6) In HPA, AE-labeled probe hybridizes to the junction of a promoter primer and a telomeric repeat. (B) The TMA/HPA procedure contains three steps: telomerase extension, amplification (TMA), and detection (HPA). The details of TMA mixture components are described in Materials and Methods.

with RNase after the telomerase elongation step (Fig. 1A, step 1). RNase (Boehringer Mannheim) was added at 0.5 mg/assay for 30 min at 37 °C to inactivate telomerase. Phenol-chloroform treatment was performed to exclude RNase activity.

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Hirose et al.: Measurement of telomerase activity

To increase TMA amplification efficiency, a tag sequence that was not complementary to the telomeric repeat sequence was added to the 39 end of the reverse primer in TMA. All experiments were performed at least three times to confirm their reproducibility.

trap assay TRAP assays were performed with the TRAPeze Telomerase Detection Kit (Oncor Inc.). In brief, the CHAPS extract was incubated with TRAP mixture including 2 U of Taq polymerase at 20 °C for 30 min and then heated to 90 °C for 3 min to inactivate the telomerase activity. The reaction mixture was subjected to 31 PCR cycles at 94 °C for 30 s and 60 °C for 30 s. TRAP reaction products were separated by 12.5% polyacrylamide gel electrophoresis and detected by staining with SYBR green (TaKaRa) (21).

hpa The basic methodology for preparation of probe was described previously (16, 17). An acridinium ester-labeled (AE-labeled) oligonucleotide, which hybridizes to the junction of a promoter primer and a telomeric repeat, was synthesized. For hybridization, 10 mL of the amplified product was diluted to 100 mL with H2O in a 12 3 75 mm polypropylene tube. One hundred microliters of the probe solution [0.1 mol/L lithium succinate buffer, pH 5.2, containing 200 g/L lauryl sulfate, 1.2 mol/L lithium chloride, 20 mmol/L EDTA, and 20 mmol/L ethylenebis(oxyethylenenitrilo)tetraacetic acid] containing 0.05 pmol of the AE-labeled probe was added. Samples were lightly vortex-mixed and incubated at 65 °C for 20 min. Differential hydrolysis of the bound vs free probe was performed by the addition of 300 mL of hydrolysis buffer (0.6 mol/L sodium tetraborate buffer, pH 8.5, 50 mL/L Triton X-100) and incubation of the sample at 65 °C for 10 min.

After the sample was cooled at room temperature for 5 min, the chemiluminescence was measured in a luminometer (Gen-Probe Leader I; Gen-Probe Inc.), using an automated reagent-injection method involving two detection reagents (reagent I: 1 mL/L H2O2 and 1 mmol/L nitric acid; reagent II: 1 mol/L NaOH). The resulting chemiluminescence was integrated for 2 s, and the results were expressed in relative light units (rlu). All of these steps were performed in a single 12 3 75 mm polypropylene tube. To extend the detection range, chemiluminescence was quenched by the addition of 2.5 pmol of unlabeled probe.

Results determination of tag length of reverse primer Addition of a tag sequence at the 59 end of a reverse primer that is not complementary to the telomeric repeat sequence should maximize the chance that PCR products correctly represent the lengths of the original telomerase products (22–25). To determine whether a tag sequence may also be beneficial in TMA, 7- to 12-bp tag sequences were added to the 59 end of the reverse primer (Fig. 2). The addition of tag sequences improved TMA efficiency, with a 10-bp tag giving the highest signal, approximately fourfold higher than the signal with no tag reverse primer. Therefore, we used a 10-bp tag reverse primer for all subsequent experiments.

sensitivity of tma/hpa The sensitivity of TMA/HPA was examined using K562 and HL60 cell extracts, which were serially diluted from 500 to 1 cell equivalent. As shown in Fig. 3, signals of 1 K562 cell equivalent and 2.5 HL60 cell equivalents were

Fig. 2. TMA amplification efficiency by tag length of reverse primer. To determine the efficiency of the tag sequence in TMA, 7- to 12-bp tag sequences were added to the 59 end of the reverse primer. Bold lowercase letters of each sequence represent tag sequence.


;100 000 rlu, which were significantly higher than that of the extract-free sample (8000 rlu). Signals of TMA/HPA were almost dose-dependent. We showed previously that TRAP/HPA could detect a single K562 cell and that the detection limit of TRAP/HPA is equivalent to or lower than that of conventional TRAP with autoradiography (15). These results indicated that the detection limit of TMA/HPA is also equivalent to, or lower than that of conventional TRAP. To determine whether the TMA/ HPA signals are dependent on telomerase activity, RNase was incubated with 500 cell equivalents of extract before the extension reaction by telomerase (Fig. 1A, step 1). After RNase exposure, the disappearance of TMA/HPA signals paralleled the decrease in the electrophoresis ladders of conventional TRAP (Fig. 3). This result confirmed that TMA amplification products come from telomerase-processed primers and that TMA/HPA signals are dependent on telomerase activity.

quantification of tma/hpa Because the AE-labeled probe can hybridize to the junction of a promoter primer and a telomeric repeat, one molecule of AE-labeled probe hybridizes to only one molecule of product (Fig. 1A, step 6). Although this should be useful for the quantification of telomerase activity, TMA/HPA signals tend to reach a plateau when samples contain high telomerase activity. To give HPA a wide linearity range, chemiluminescence was quenched by the addition of 2.5 pmol of unlabeled probe (26). As shown in Fig. 4, TMA/HPA has a linearity range from 1 to 1000 cell equivalents by addition of unlabeled probe.

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telomerase activity in tissues The presence of inhibitors of the conventional TRAP assay has been reported (13, 14). To determine the effect of these inhibitors on TMA/HPA, 10 clinical samples that contained inhibitors for the conventional TRAP assay were used in TMA/HPA, and then the phenol-chloroform treatment was performed after the telomerase extension step (Figs. 5 and 6). In the conventional TRAP assay, protein exclusion from extracts by the phenol-chloroform treatment clearly increased the number of 6-base ladders in electrophoresis. The same result was achieved even if the extract was diluted and the equivalent of 1 mg of protein was used (Fig. 5). In contrast, TMA/HPA signals remained unchanged by the phenol-chloroform treatment when the equivalent of 1 mg of protein was used (Fig. 5). This phenomenon was confirmed with another clinical sample, which was diluted from 5 to 0.01 mg of protein (Fig. 6). When ,1 mg of protein was used, TMA/HPA signals without phenol-chloroform treatment were identical to those with treatment; however, there was a marked difference in ladder extension in the conventional TRAP. The same result was obtained with seven other colon samples (data not shown). Furthermore, TMA/ HPA could generate positive signals when 0.01 mg of protein was used, whereas no ladder was observed with concentrations of protein as low as 0.01 mg. These data demonstrated that when low concentrations of protein were added, TMA/HPA was minimally influenced by the inhibitors in tissue samples compared with the conventional TRAP assay.

Fig. 3. Sensitivity of TMA/HPA. Extracts from K562 and HL60 cells were used. Serial dilutions of extracts equivalent to 0 –500 cells were processed for TMA/HPA. The differences between TMA/HPA signals with (1) or without (2) RNase were compared with the 500-cell equivalent extract.

Fig. 4. Quantification of TMA/HPA. Cell extracts equivalent to 0 –1000 K562 cells were used. Unlabeled probe was added in a 50-fold excess over AE-labeled probe for the quantification.

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Fig. 5. Effect of inhibitors in CRC11 and CRC19 on TMA/HPA. Extracts from colon cancer samples CRC11 and CRC19 were diluted and applied to TMA/HPA and TRAP. The phenol-chloroform treatment was performed after the extension step by telomerase. Dilutions containing 1 or 5 mg of protein from CRC 11 and CRC 19 were used in each assay.

The results of TMA/HPA of liver samples (Fig. 7) confirmed the previous results from conventional TRAP and TRAP/HPA and showed that TMA/HPA could quantitatively detect telomerase activity more easily than conventional TRAP when the cutoff value was calculated as 2 SD above the mean activity of non-tumor liver tissues. The calculated cutoff values with 1 and 0.1 mg of protein were 30 841 rlu and 11 891 rlu, respectively. Poorly differentiated HCCs had higher telomerase activity than well or moderately differentiated HCCs. Because TMA/HPA has a detection limit equivalent to or lower than conventional TRAP, telomerase activity was positive in 27 of 33 HCCs (81.8%) when both 1 and 0.1 mg of protein were used. In contrast, among 44 non-tumor lesions, only 3 samples with 1 mg of protein and 2 samples with 0.1 mg of protein were above the cutoff value. Although two samples of non-tumor lesions were above the cutoff value when 0.1 mg of protein was used (Fig. 7B), it was easier to discriminate between HCC samples and non-tumor lesions than with 1 mg of protein (Fig. 7A). Moreover, because TMA/HPA has a detection limit equivalent to or lower than conventional TRAP, it gave a high positive rate (81.8%) even when 0.1 mg of protein was used.

Fig. 6. Effect of inhibitors in CRC 1 on TMA/HPA. The extract from colon cancer sample CRC1 was diluted and applied to TMA/HPA and TRAP. The phenol-chloroform treatment was performed after the extension step by telomerase. Serially diluted CRC 1 extract containing 0.01–5 mg of protein was used in the assay.

Discussion The development of the TRAP assay has opened the door for the detection and measurement of telomerase activity. This technique can detect telomerase activity in most tumor samples. In the future, it is expected that telomerase will become more important as a biomarker for the diagnosis and prognosis of cancer. In an effort to make these assays suitable for routine clinical use, several modifications have been required (22–25). An internal control was used to detect inhibitors in clinical samples and to calibrate telomerase activity, and a tag sequence was added to the 59 end of the reverse primer to maximize the chance for PCR products to represent the lengths of the original telomerase products. However, the detection step of the TRAP assay, which requires electrophoresis, is still inconvenient for clinical use because it cannot be applied to large numbers of clinical samples. We have developed a novel TMA/HPA assay to detect and measure telomerase activity. TMA is an isothermal amplification method and does not require any thermocycling. Another advantage of TMA is that the primary products of amplification are RNA. These RNA products are very labile outside the reaction tube, thereby reducing the risk of laboratory contamination and false-positive results. In addition, RNA amplicons do not require strand


Fig. 7. Telomerase activity in 1 mg (A) and 0.1 mg (B) of protein from liver tissues. The vertical line is the cutoff value (2 SD above the mean for non-tumor liver tissue).

separation before hybridization with a detection probe. TMA and HPA are already used together in commercially available diagnostic kits for the detection microorganisms such as Chlamydia and Mycobacterium tuberculosis (GenProbe Inc.). These technologies have been demonstrated as suitable for routine clinical laboratory use. To apply TMA to the amplification of products extended by telomerase, two primers used in TMA were modified. A T7 RNA polymerase promoter sequence was added to the 59 end of the sequence to provide a substrate for telomerase, and it gave equivalent results to those of the TS primer used in the TRAP assay (data not shown). Furthermore, the reverse primer modified by the addition of a tag sequence that is not complementary to the telomeric sequence increased the amplification efficiency of TMA (Fig. 2). A tag sequence is known to increase the specificity of amplification in TRAP (23, 24), and the addition of a tag sequence may also increase the hybridization of reverse primer to the first-round products in TMA. HPA was used to detect TMA amplification products to measure telomerase activity. HPA is a homogeneous and very fast assay that can be completed in ,1 h. Because HPA results are automatically expressed in rlu by a luminometer, results are easy to quantify. TMA/HPA could detect the telomerase activity of 1 or 2.5 cell equivalents extracted from cell lines (Fig. 3). The detection limit of TMA/HPA was equivalent or better to that of conventional TRAP. Moreover, because the addition of cold probe facilitated the sample quantification by TMA/ HPA in the range of 1 to 1000 cell equivalents (Fig. 4), TMA/HPA was considered to be appropriate for measuring telomerase activity in clinical samples. The TMA/ HPA assay format is not technically demanding; therefore, it is applicable to a high-throughput format and

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could be applied to a large number of clinical samples at the same time. Researchers have reported independently that some clinical samples contain Taq polymerase inhibitors and that the addition of an internal control in TRAP was an efficient way to prevent false-negative results caused by the presence of these inhibitors (13, 14). However, it remains difficult in TRAP to measure the area or intensity of bands on gels by densitometry for quantitative analysis, and this approach cannot quantify telomerase activity in clinical samples. Therefore, we investigated the effect of inhibitors in clinical samples on TMA/HPA (Figs. 5 and 6). Extracts from colon cancer, which have been shown to include inhibitors for TRAP, were used. TMA/HPA results were also influenced by inhibitor when 5 mg of protein was used (Figs. 5 and 6). However, when extracts were diluted and #1 mg of protein was used, TMA/HPA signals without phenol-chloroform treatment were equivalent to those with phenol-chloroform treatment. Because those results were obtained with 10 colon samples, they indicate that TMA/HPA may be not influenced by inhibitors when 1 mg of protein is used. The conventional TRAP was influenced by inhibitors even in diluted extracts. We have demonstrated that TMA/HPA minimizes the influence of inhibitors in diluted clinical samples with lower protein concentrations. Tissue samples from liver were used to verify the detection limit of TMA/HPA and the influence of the inhibitors for TMA/HPA (Fig. 7). Results confirmed not only the previous results (15) but also the sensitivity of TMA/HPA. The positive rate was 81.8% (27 of 33) when either 1 or 0.1 mg of protein was used. Moreover, although signals of non-tumor lesions were frequently higher than those for healthy tissue, only a few non-tumor lesion samples had higher signals than the cutoff value when the cutoff value was calculated as 2 SD above the mean signal of all non-tumor lesions. The weak signals of non-tumor lesions may be caused by a small number of hidden HCC cells, a small number of precancerous cells, regenerating nondiseased liver cells, or infiltrating active lymphocytes. In general, because liver tissues contained less inhibitory activity than colon tissues (12, 20), TMA/HPA was not influenced by inhibitory activity when 1 mg of protein from liver tissue extract was used. To monitor the inhibitory activity of clinical samples in TMA/HPA, we needed to use a dual kinetic assay (27), which can distinguish between the chemiluminescence of telomerase products and that of internal control products in one tube. Although inhibitor problems may not be resolved completely in the current method, TMA/HPA is rapid, can be completed in ,4 h, has a low detection limit and a wide linearity range, and is therefore practical for a large number of clinical assays. This new method for measuring telomerase activity should be very useful for the diagnosis of a variety of cancers.

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We thank Yukio Matsuoka and Keiichi Kamisango at Chugai Diagnostics Science Co., Ltd. for encouragement and helpful suggestions, and Richard Harvey at GenProbe Inc. for critical reading. We also thank Toshio Nakanishi and Mikiya Kitamoto, First Department of Internal Medicine, Hiroshima University School of Medicine, for liver samples.

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