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Expression of Telomerase Activity, Human Telomerase RNA, and Telomerase Reverse Transcriptase in Gastric Adenocarcinomas Jinyoung Yoo, M.D., Ph.D., Sonya Y

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Expression of Telomerase Activity, Human Telomerase RNA, and Telomerase Reverse Transcriptase in Gastric Adenocarcinomas Jinyoung Yoo, M.D., Ph.D., Sonya Y Expression of Telomerase Activity, Human Telomerase RNA, and Telomerase Reverse Transcriptase in Gastric Adenocarcinomas Jinyoung Yoo, M.D., Ph.D., Sonya Y. Park, Seok Jin Kang, M.D., Ph.D., Byung Kee Kim, M.D., Ph.D., Sang In Shim, M.D., Ph.D., Chang Suk Kang, M.D., Ph.D. Department of Pathology, St. Vincent’s Hospital, Catholic University, Suwon, South Korea esis of gastric cancer and may reflect, along with Telomerase is an RNA-dependent DNA polymerase enhanced hTR, the malignant potential of the tu- that synthesizes TTAGGG telomeric DNA onto chro- mor. It is noteworthy that methacarn-fixed tissue mosome ends to compensate for sequence loss dur- cannot as yet substitute for the frozen section in the ing DNA replication. It has been detected in 85–90% TRAP assay. of all primary human cancers, implicating that the telomerase seems to be reactivated in tumors and KEY WORDS: hTR, Stomach cancer, Telomerase, that such activity may play a role in the tumorigenic TERT. process. The purpose of this study was to evaluate Mod Pathol 2003;16(7):700–707 telomerase activity, human telomerase RNA (hTR), and telomerase reverse transcriptase (TERT) in Recent studies of stomach cancer have been di- stomach cancer and to determine their potential rected toward gaining a better understanding of relationships to clinicopathologic parameters. Fro- tumor biology. Molecular analysis has suggested zen and corresponding methacarn-fixed paraffin- that alterations in the structures and functions of embedded tissue samples were obtained from 51 oncogenes and tumor suppressor genes, genetic patients with gastric adenocarcinoma and analyzed instability, as well as the acquisition of cell immor- for telomerase activity by using a TRAPeze ELISA tality may be of relevance in the pathogenesis of kit. Tissue sections of all the samples were further these tumors (1–3). Telomerase activation is be- investigated for hTR and TERT by in situ hybridiza- lieved to be crucial in most immortal cells and tion and a sensitive immunohistochemical tech- cancer cells (4, 5); however, its clinicopathologic nique, respectively. Telomerase activity was de- significance in gastric cancer and the details of the tected in 37 (73%) tumors. Telomerase positivity mechanisms regulating telomerase activity remain from methacarn-fixed paraffin blocks was found to to be clarified. be 35% of that from frozen tissues. hTR was over- Eukaryotic chromosomes are capped with repet- expressed in 46 (90%) samples: 33/37 (89%) with itive telomere sequences that protect the chromo- and 13/14 (93%) without telomerase activation. Ex- some ends against exonucleases and ligases, thus pression of TERT was demonstrated in 40 (78%) preventing fusion, recombination, and degradation cases: 30/37 (81%) with and 10/14 (71%) without (6, 7). In human chromosomes, telomeres consist of telomerase. Telomerase activity correlated well with thousands of copies of the nucleotide sequence -and tumor differentia (037. ؍ depth of invasion (P 5'-TTAGGG-3' ranging from 5–20 kb in length. Telo- whereas hTR significantly correlated ,(022. ؍ tion (P and tumor size (P meres shorten in somatic cells with successive cell (047. ؍ with nodal metastasis (P -These data suggest that reactivated telom- divisions because of the inability of the DNA poly .(023. ؍ erase may play a significant role in the tumorigen- merase complex to replicate the 5' end of the lag- ging strand (8, 9). Germline cells compensate for this end-replication problem by expression of the enzyme telomerase, which is an RNA-dependent Copyright © 2003 by The United States and Canadian Academy of Pathology, Inc. DNA polymerase that synthesizes telomeric repeats VOL. 16, NO. 7, P. 700, 2003 Printed in the U.S.A. and incorporates them onto the 3' end of existing Date of acceptance: May 1, 2003. Supported by a grant from Samkwang Research Laboratories. telomeres (10). Telomerase activity has been iden- Address reprint requests to: Seok Jin Kang, M.D., Ph.D., Department of tified in immortal cells, cancer cells, and germ cells, Pathology, St. Vincent’s Hospital, Catholic University, Suwon, Kyungkido, South Korea 442-723; fax: 8231-244-6786; e-mail: [email protected]. but not in normal somatic cells (11–20). The reac- DOI: 10.1097/01.MP.0000077517.44687.B6 tivation of telomerase thus appears to be associated 700 with unlimited replicative potential, resulting in the (60° C) three times for 1 hour. Additional tissue immortalization of cells. Human telomerase RNA specimens were fixed in 10% phosphate-buffered (hTR) is one of the major subunits of telomerase, neutral formalin, routinely processed, and stained encompassing 11 nucleotides (5'-CUAACCCUAAC) with hematoxylin and eosin for the final diagnosis. complementary to the human telomeric repeats All of the histologic sections were reviewed and (21). Telomere shortening and eventual cell death categorized according to International Union were observed in yeast cells that were deleted for Against Cancer tumor-node-metastasis (UICC- the telomerase RNA component (22, 23). Other an- TNM) classification. Ten samples obtained from tisense experiments with hTR indicated that telom- adjacent normal tissues were run in parallel for erase inhibition may lead to telomere shortening detection of telomerase activity. and cell death in human tumor cell lines (21). Ex- pression of telomerase reverse transcriptase (TERT), a catalytic subunit component, has been Telomerase Assay reported in telomerase-positive cell lines but not in Telomerase assay was performed as described telomerase-negative primary fibroblasts (24, 25), previously. Each frozen sample was thawed to Ϫ20° yet the correlation between telomerase activity and C and cryosectioned using a Cryostat apparatus expression of hTR and TERT has not been well (Leica, Inc., Deerfield, IL). Ten consecutive 10-␮m documented in clinical samples. sections were obtained and suspended in ice-cold To investigate the expression of telomerase, hTR, 1ϫ CHAPS lysis buffer (10 mM Tris-HCl, pH 7.5, 1 and TERT in stomach cancer and to determine their mM MgCl2,1mM EGTA, 0.1 mM benzamidine, 5 mM potential relationships with clinicopathologic pa- ␤-mercaptoethanol, 0.5% CHAPS, and 10% glyc- rameters, we analyzed 51 samples for telomerase erol) containing RNase inhibitor at a final concen- activity, hTR, and TERT with the use of a telomerase tration of 100–200 U/mL. After 30 minutes of incu- (TRAP) assay, in situ hybridization (ISH), and im- bation on ice, the lysate was centrifuged at 14,000 ϫ munohistochemistry, respectively. g for 25 minutes at 4° C, and the supernatant was The TRAP is a sensitive and efficient polymerase transferred into a fresh tube. Protein concentra- chain reaction (PCR)–based telomerase activity de- tions of the tissue extract were measured by the tection method (4, 26, 27), but its limitation is the Bradford method (Bio-Rad, Hercules, CA), and an need for either fresh or frozen tissues for the reac- aliquot of extract containing 6 ␮g of protein was tion. Methacarn-fixed paraffin-embedded tissues used for each TRAP assay. For RNA quality control, were found to be suitable for molecular analysis the lysate (total, 60 ␮g of protein extract) was mixed (28–30). Methacarn fixation is recommended as a with TRIzol (GIBCO-BRL, Gaithersburg, MD) and valuable approach for routine application, consid- chloroform and then centrifuged. RNA was precip- ering its advantages for molecular studies at the itated from the aqueous phase by the addition of mRNA level and for immunohistochemical detec- isopropanol, washed and dissolved in RNase-free tion of proteins. To determine whether methacarn- water. RNA was electrophoresed in formamide-1% fixed paraffin-embedded tissues can replace cryo- agarose gel. The remaining extract was immediately stat sections for the TRAP assay, duplicate studies stored frozen at Ϫ80° C. were performed using both frozen and matched For the TRAP assay, TRAPeze ELISA (Introgen methacarn-fixed specimens. Co., Purchase, NY), a telomerase detection kit, was used according to the manufacturer’s instructions with minor modifications. Fifty ␮L of reaction mix- ␮ ␮ ϫ MATERIAL AND METHODS ture containing 1 g of protein extract, 10 Lof5 TRAP reaction mix (Tris buffer, primers, biotinyl- A total of 51 stomach cancer tissues were ob- ated TS primer and RP primer, dNTPs and DNP- tained from patients who underwent gastrectomy dCTP, and oligomer mix for amplification of 36-bp at Catholic University St. Vincent’s Hospital, South internal control band), and 2 units of Taq DNA Korea. Upon surgical removal, tumor tissues were polymerase (Life Technologies, Gaithersburg, MD), immediately frozen in liquid nitrogen and stored at was incubated for 30 minutes at 30° C and subse- Ϫ80° C until they were subjected to TRAP assay. To quently subjected to two-step PCR at 94° C for 30 compare unfixed frozen tissue samples with seconds and 55° C for 30 seconds for 33 cycles. methacarn-fixed tissues, a piece of the specimen Analysis of each sample consisted of three assays: from the same area taken for the frozen section one with a test extract of 1 ␮g, one with 0.2 ␮gof used as TRAP assay was fixed in methacarn for 1 protein, and one with a heat-inactivated lysate at hour at 4° C, dehydrated three times for 1 hour each 85° C for 10 minutes before the assay. For a primer- in fresh 100% ethanol at 4° C, placed in xylene once dimer/PCR contamination control, 2 ␮Lof1ϫ for 1 hour and then three times for 30 minutes at CHAPS lysis buffer was substituted for the extract. room temperature, and immersed in hot paraffin Each set of experiments also included telomerase- Telomerase in Gastric Cancer (J. Yoo et al.) 701 positive control cell extract and PCR/ELISA-positive dure detected the probe-target hybrid. This in- control supplied in the kit. Nonradioactive detec- cluded incubating slides with the sequential addi- tion of the telomerase products was performed by tion of mouse anti-fluorescein antibody, ELISA protocol.
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