Skip to main content
SpringerLink
Log in

Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP)

  • Genetics — Cellular Immortality
  • Published:
Methods in Cell Science

Abstract

The association of human telomerase activity with an indefinite replicative capacity of cells in vitro and advanced tumors in vivo is gaining wide support. The increasing interest in studying various aspects of telomerase expression in cancer required the development of a sensitive and reliable protocol for the extraction and detection of telomerase activity in cell culture material, and from small tissue samples obtained from biopsy, surgical reaction of tumors, and autopsy. Recently a novel procedure for the extraction and detection of telomerase activity was developed (Science 1994; 266: 2011–2015) which resulted in an estimated 104 fold improvement in detectability compared with previous methods. The described procedures not only dramatically increased sensitivity but also allowed fast and efficient detection of telomerase activity in a large number of samples. A number of technical aspects which are of eritical importance for reproducibility and reliability of this assay using clinical material are addressed in this report. In addition, new methods to perform telomerase assays without the use of radioisotopes are described.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

AEBSF:

4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochlorine

BCA:

bieinchoninic acid

CCD camera:

charged coupled device camera

CHAPS:

3-[(3-cholamidopropyl)-dimethyl-ammonio]-l-propanesulfonate

DEPC:

diethyl pyrocarbonate

DPBS:

Dulbecco's phosphate buffered saline

EGTA:

ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid

HPLC:

high pressure liquid chromatography

HPV:

human papillomavirus

PDA:

piperazine diacrylamide

PCR:

polymerase chain reaction

T4g32 protein:

T4 gene 32 protein

TRAP:

telomeric repeat amplification protocol

TRF:

terminal restriction fragment

References

  1. Hara E, Tsuruni H, Shinozaki A, Nakada S, Oda K (1991). Cooperative effect of antisense-Rb and antisense-p53 oligomers on the extension of life span in human diploid fibroblasts, TIG-1. Biochim Biophys Res Commun 179: 528–534.

    Google Scholar 

  2. Huschtscha LL, Holliday R (1983). Limited and unlimited growth of SV40-transformed cells from human diploid MRC-5 fibroblasts. J Cell Sci 63: 77–99.

    Google Scholar 

  3. Linder S, Marshall H (1990). Immortalization of primary cells by DNA tumor viruses. Exp Cell Res 191: 1–7.

    Google Scholar 

  4. Shay JW, Tomlinson G, Piatyszek MA, Gollahon LS (1995). Spontaneous in vitro immortalization of breast epithelial cells from a patient with Li-Fraumeni syndrome. Mol Cell Biol 15: 425–432.

    Google Scholar 

  5. Wright WE, Pereira-Smith OM, Shay JW (1989). Reversible cellular senescence: Implications for a two-stage model for the immortalization of normal human diploid fibroblasts. Mol Cell Biol 9: 3088–3092.

    Google Scholar 

  6. Harris CC (1987). Human tissues and cells in carcinogenesis research. Cancer Res 47: 1–10.

    Google Scholar 

  7. Shay JW, Wright WE, Werbin H (1991). Defining the molecular mechanisms of human cell immortalization. Biochem Biophys Acta 1072: 1–7.

    Google Scholar 

  8. Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, Greider CW, Harley CB (1992). Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci (USA) 89: 10114–10118.

    Google Scholar 

  9. Counter CM, Avillon AA, LeFeuvre CE, et al. (1992). Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J 11: 1921–1929.

    Google Scholar 

  10. Counter CM, Botelho FM, Wang P, Harley CB, Bacchetti S (1994). Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes. J Virol 68: 3410–3414.

    Google Scholar 

  11. Counter CM, Hirte HW, Bacchetti S, Harley CB (1994). Telomerase activity in human ovarian carcinoma. Proc Natl Acad Sci (USA) 91: 2900–2904.

    Google Scholar 

  12. Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC (1990). Telomere reduction in human colorectal carcinoma and with ageing. Nature 346: 866–868.

    Google Scholar 

  13. Vaziri H. Schächter F, Uchida I, Wei L, Zhu X, Effors R, Cohen D, Harley CB (1993). Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Am J Hum Genet 52: 661–667.

    Google Scholar 

  14. Wright WE, Shay JW (1992). Telomere positional effects and the regulation of cellular senescence. Trends Genet 8: 193–197.

    Google Scholar 

  15. Blackburn EH (1994). Telomeres: No end in sight. Cell 77: 621–623.

    Google Scholar 

  16. Olovnikov AM (1973). A theory of marginotomy. J Theor Biol 41: 81–190.

    Google Scholar 

  17. Watson JD (1972). Origin of concatameric T4 DNA. Nature 239: 197–201.

    Google Scholar 

  18. Shay JW, Werbin H, Wright WE (1994). Telomere shortening may contribute to aging and cancer: A perspective. Mol Cell Dif 2: 1–21.

    Google Scholar 

  19. Shay JW, Wright WE, Werbin H (1993). Loss of telomeric DNA during aging may predispose cells to cancer. Int J Oncology 3: 559–563.

    Google Scholar 

  20. Shay JW, Pereira-Smith OM, Wright WE (1991). A role for both Rb and p53 in the regulation of human cellular senescence. Exp Cell Res 196: 33–39.

    Google Scholar 

  21. Adamson DJ, King DJ, Haites NE (1992). Significant telomere shortening in childhood leukemia. Cancer Genet Cytogenet 61: 204–206.

    Google Scholar 

  22. Hiyama E, Yokoyama T, Hiyama K, Yamakido M, Santo T, Kodama T, Ichikawa T, Matsuura Y (1995). Alteration of telomeric repeat length in adult and childhood solid neoplasias. Int J Oncology 6: 13–16.

    Google Scholar 

  23. Mehle C, Ljungberg B, Roos G (1994). Telomere shortening in renal cell carcinoma. Cancer Res 54: 236–241.

    Google Scholar 

  24. Odagiri E, Kanda N, Jibiki K, Demura R, Aikawa E, Demura H (1994). Reduction of telomeric length and c-erbB-2 gene amplification in human breast cancer, fibroadenoma, and gynecomastia. Relationship to histologic grade and clinical parameters. Cancer 73: 2978–2984.

    Google Scholar 

  25. Smith JK, Yeh G (1992). Telomere reduction in endometrial adenocarcinoma. Am J Obstet Gynecol 167: 1883–1887.

    Google Scholar 

  26. deLange T, Shiue L, Myers RM, Cox DR, Naylor SL, Killery AM, Varmus HE (1990). Structure and variability of human chromosome ends. Mol Cell Biol 10: 518–527.

    Google Scholar 

  27. Greider CW, Blackburn EH (1985). Identification of a specific telomere terminal transferase activity inTetrahymena extracts. Cell 43: 405–413.

    Google Scholar 

  28. Greider CW, Blackburn EH (1989). A telomeric sequence in the RNA ofTetrahymena telomerase required for telomere repeat synthesis. Nature 337: 331–337.

    Google Scholar 

  29. Morin GB (1989). The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59: 521–529.

    Google Scholar 

  30. Hiyama E, Hiyama K, Yokoyama T, Matsuura Y, Piatyszek MA, Shay JW (1995). Correlating telomerase activity levels with human neuroblastoma outcomes. Nature Medicine 1: 249–255.

    Google Scholar 

  31. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PLC, Coviello GM, Wright WE, Weinrich SL, Shay JW (1994). Specific association of human telomerase activity with immortal cells and cancer. Science 266: 2011–2015.

    Google Scholar 

  32. Nilsson P, Mehle C, Remes K, Ross G (1994). Telomerase activity in vivo in human malignant hematopoietic cells. Oncogene 9: 3043–3048.

    Google Scholar 

  33. Shay JW, Brasiskyté D, Ouellette M, Piatyszek MA, Werbin H, Ying Y, Wright WE (1994). Analysis of telomerase and telomeres. In Adolph KW (ed), Gene and chromosome analysis. San Diego: Academic Press, 5 part C: 263–280.

    Google Scholar 

  34. Hiyama K, Hiyama E, Ishioka S, Yamakido M, Inai K, Gazdar AF, Piatyszek MA, Shay JW (1995). Telomerase activity in human lung cance: Do all lung cancers consist of immortal cells? AACR abstracts 1995, No. 3299.

  35. Mehle C, Piatyszek MA, Ljungberg B, Shay JW, Roos G (1995). Telomerase activity and clinical correlations in human renal cell carcinoma. AACR abstracts 1995, No. 3309.

  36. Sommerfeld HJ, Meeker AK, Piatyszek MA, Shay JW, Coffey DS (1995). Telomere shortening and telomerase activity in human prostate tissue. AACR abstracts 1995, No. 3311.

  37. Morin GB (1991). Recognition of chromosome truncation site associated with α-thalassaemia by human telomerase. Nature 353: 454–456.

    Google Scholar 

  38. Kaczorowski T, Furmanek B, Sektas MA (1994). Method for removal of radioactive nucleotides from electrophoretic buffers. BioTechniques 16: 1030–1031.

    Google Scholar 

  39. Bassam BJ, Caetano-Anollés G, Gresshoff PM (1991). Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 196: 80–83.

    Google Scholar 

  40. Hsu IC, Metcalf RA, Sun T, Welsh JA, Wang NJ, Harris CC (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature 350: 427–428.

    Google Scholar 

  41. Fernley HN (1971). Mammalian alkaline phosphatases. In: Boyer PD (ed), The enzyntes. New York/London: Academic Press, vol 4, pp 417–447.

    Google Scholar 

  42. Posen S, Neale FC, Clubb JS (1995). Heat inactivation in the study of human alkaline phosphatases. Ann Int Med 62: 1234–1243.

    Google Scholar 

  43. Akane A, Matsubara K, Nakamura H, Takahashi S, Kimura K (1994). Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains: A major inhibitor of polymerase chain reaction (PCR) amplification. J Forensic Sci 39: 362–372.

    Google Scholar 

  44. Niederhauser C, Höfelein C, Wegmüller B, Lüthy J, Candrian U (1994). Reliability of PCR decontamination systems. PCR Meth Appl 4: 117–123.

    Google Scholar 

  45. Chiu C-P, Dragowski V. Kim NW, Thomas TE, Harley CB, Lansdorp PM (1995). Telomerase activity in hematopoietic progenitor cell extracts from adult bone marrow. AACR abstracts 1995, No. 3306.

  46. Shay JW, Wright WE, Brasiskyté D, Van der Haegen BA (1993). E6 of human papilloma virus 16 can overcome the M1 stage of immortalization in human mammary epithelial cells but not in human fibroblasts. Oncogene 8: 1407–1413.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Cell Biology and Neurosciences, University of Texas Southwestern Medieal Center at Dallas, 5323 Harry Hines Boulevard, 75235-9039, Dallas, TX, USA

    Mieczyslaw A. Piatyszek, Woodring E. Wright & Jerry W. Shay PhD

  2. Geron Corporation, Menlo Park, California, USA

    Nam W. Kim & Scott L. Weinrich

  3. Sacond Department of Internal Medicine, Hiroshima University School of Medicine, Hiroshima, Japan

    Keiko Hiyama

  4. Department of General Medicine, Hiroshima University School of Medicine, Hiroshima, Japan

    Eiso Hiyama

Authors
  1. Mieczyslaw A. Piatyszek
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nam W. Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Scott L. Weinrich
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Keiko Hiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Eiso Hiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Woodring E. Wright
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jerry W. Shay PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jerry W. Shay PhD.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Piatyszek, M.A., Kim, N.W., Weinrich, S.L. et al. Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP). Methods Cell Sci 17, 1–15 (1995). https://doi.org/10.1007/BF00981880

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00981880

Key words

Search

Navigation