Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1998-12-23
2001-04-03
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S006120, C435S193000
Reexamination Certificate
active
06210915
ABSTRACT:
TECHNICAL FIELD
The present invention relates to telomerase, a ribonucleoprotein enzyme involved in telomere DNA synthesis, and provides protocols for extracting telomerase from cell samples and assaying the extracted telomerase. The invention provides methods and compositions relating to the fields of molecular biology, and medical diagnostic and prognostic technologies.
BACKGROUND
Telomeres are genetic elements located at the ends of all eukaryotic chromosomes which preserve genome stability and cell viability by preventing aberrant recombination and degradation of DNA (McClintock, 1941; Muller, 1938). In humans, the telomeric sequence is composed of 10-20 kilobases of TTAGGG repeats (Blackburn, 1991; de Lange et al., 1990). There is increasing evidence that gradual loss of telomeric repeat sequences may be a timing (“clock”) mechanism limiting the number of cellular divisions in normal cells (Allsopp et al., 1992; Harley et al., 1990; Hastie et al., 1990; Vaziri et al., 1993). In contrast, immortal cells are capable of maintaining a stable telomere length by upregulating or reactivating telomerase, a ribonucleoprotein enzyme that is able to add TTAGGG repeats to the ends of chromosomes (Greider and Blackburn, 1985; Greider and Blackburn, 1989; Morin, 1989).
Methods for detecting telomerase activity, as well as for identifying compounds that regulate or affect telomerase activity, together with methods for therapy or diagnosis of cellular senescence and immortalization by controlling or measuring telomere length and telomerase activity, have been described. See PCT patent publication No. 93/23572, U.S. Pat. Nos. 5,629,154, 5,648,215, 5,645,986, 5,695,932 and 5,489,508. Each of the foregoing patent publications are incorporated herein by reference.
For example, U.S. Pat. Nos. 5,629,154 and 5,648,215 describe in detail the preparation of a cell extract using a detergent lysis method and the analysis of telomerase activity by the Telomeric Repeat Amplification Protocol (TRAP assay). The telomerase activity assays described therein involve the extension of a nucleic acid substrate by telomerase and replication of extended substrates in a primer extension reaction, such as the polymerase chain reaction (PCR).
Other telomerase extraction methods use hypotonic swelling and physical disruption of cells and telomerase activity is assayed using an oligonucleotide substrate, a radioactive deoxyribonucleoside triphosphate (dNTP) for labelling any telomerase-extended substrate, and gel electrophoresis for resolution and display of products (Morin, 1989). Because telomerase stalls and can release the DNA after adding the first G in the 5′-TTAGGG-3′ telomeric repeat, the characteristic pattern of products on the gel is a six nucleotide ladder of extended oligonucleotide substrates. The phase of the repeats depends on the 3′-end sequence of the substrate; telomerase recognizes where the end is in the repeat and synthesizes accordingly to yield contiguous repeat sequences.
Using the TRAP assay, telomerase activity has been detected in 85% of primary human tumors tested from a variety of tissue types (Kim et al., 1994; Shay and Bacchetti, 1997). The detection of telomerase activity in human cells almost always correlates with indefinite proliferation capability (immortalization). U.S. Pat. No. 5,648,215 describes the presence of telomerase activity in somatic cells as indicative of the presence of immortal cells, such as certain types of cancer cells, which can be used to make that determination even when the cells would be classified as non-cancerous by pathology.
While most published reports to date only indicate that telomerase activity is present or absent, methods for cancer prognosis based upon detecting telomerase activity levels have also been described. See U.S. Pat. Nos. 5,639,613 and 5,693,474, and U.S. application Ser. No. 08/485,454 entitled “Telomerase Activity Associated with Hematological and Colorectal Malignancies” filed Jun. 7, 1995.
It has become increasingly evident that accurately quantitating telomerase activity levels in cell samples would be useful in the elucidation of the biological mechanisms by which telomerase acts, as well as for patient diagnosis and prognosis based on telomerase activity levels. Although useful information has been obtained from telomerase assays using the detergent based extraction procedure described above, this invention provides an improved telomerase extraction procedure that allows a more accurate determination of telomerase activity levels in a cell sample.
Relevant Literature
Allsopp, R. C., Vaziri, H., Patterson, C., Goldstein, S., Younglai, E. V., Futcher, A. B., Greider, C. W., and Harley, C. B. (1992). Telomere length predicts replicative capacity of human fibroblasts. PNAS 89, 10114-10118.
Blackburn, E. H. (1991). Structure and function of telomeres. Nature 350, 569-573.
Cremo, C. R., Herron, G. S., and Schimerlik, M. I. (1981). Solubilization of the atrial muscarinic acetylcholine receptor: a new detergent system and rapid assays. Anal. Biochem. 115, 331-338.
de Lange, T., Shiue, L., Myers, R. M., Cox, D. R., Naylor, S. L., Killery, A. M., and Varmus, H. E. (1990). Structure and variability of human chromosome ends. Mol. Cell. Biol. 10, 518-526.
Gollahon, L. S., and Shay, J. W. (1996). Immortalization of human mammary epithelial cells transfected with mutant p53 (273his). Oncogene 12, 715-725.
Greider, C. W., and Blackburn, E. H. (1985). Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405-413.
Greider, C. W., and Blackburn, E. H. (1989). A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 337, 331-337.
Harley, C. B., Fletcher, A. B., and Greider, C. W. (1990). Telomeres shorten during aging. Nature 345, 458-460.
Hastie, N. D., Dempster, M., Dunlop, M. G., Thompson, A. M., Green, D. K., and Allshire, R. C. (1990). Telomere reduction in human colorectal carcinoma and with ageing. Nature 346, 866-868.
Holt, S. E., Norton, J. C., Wright, W. E., and Shay, J. W. (1996). Comparison of the telomeric repeat amplification protocol (TRAP) to the new TRAP-eze telomerase detection kit. Methods in Cell Science 18, 237-248.
Holt, S. E., Shay, J. W., and Wright, W. E. (1996). Refining the telomere-telomerase hypothesis of aging and cancer. Nature Biotech. 14, 836-839.
Holt, S. E., Wright, W. E., and Shay, J. W. (1996). Regulation of telomerase activity in immortal cell lines. Mol. Cell. Biol. 16, 2932-2939.
Ignatoski, K. M., and Verderame, M. F. (1996). Lysis buffer composition dramatically affects extraction of phosphotyrosine-containing proteins. Biotechniques 20, 794-6.
Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D., Ho, P. L., Coviello, G. M., Wright, W. E., Weinrich, S. L., and Shay, J. W. (1994). Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011-2015.
McClintock, B. (1941). The Stability of Broken Ends of Chromosomes in Zea maize. Genetics 26, 234-282.
Morin, G. B. (1989). The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59, 521-529.
Muller, H. J. (1938). The Remaking of Chromosomes. The Collecting Net 13, 181-198.
Piatyszek, M. A., Kim, N. W., Weinrich, S. L., Keiko, H., Hiyama, E., Wright, W. E., and Shay, J. W. (1995). Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP). Methods in Cell Science 17, 1-15.
Shay, J. W., and Bacchetti, S. (1997). A survey of telomerase activity in human cancer. European Journal of Cancer 5, 787-791.
Vaziri, H., Schachter, F., Uchida, I., Wei, L., Zhu, X., Effros, R., Cohen, D., and Harley, C. B. (1993). Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Amer. J. Human Genetics 52, 661-667.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for the efficient extraction of telomerase from a cell sample.
It is a further object of the p
Norton, Jr. James C.
Shay Jerry W.
Wright Woodring E.
Banait Narinder S.
Lillis Marcella
University of Texas Systems
Weber Jon P.
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