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ABSTRACT: During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization, the selection of rare, spontaneously arising immortalized cells, or the use of an entire viral genome. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT) in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.
Nature 08/1999; 400(6743):464-8. · 36.28 Impact Factor
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ABSTRACT: The immortalization of human cells is a critical step during tumorigenesis. In vitro, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization.
Proceedings of the National Academy of Sciences 01/1999; 95(25):14723-8. · 9.68 Impact Factor
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ABSTRACT: Activation of telomerase, the enzyme that synthesizes the telomere ends of linear chromosomes, has been implicated in human cell immortalization and cancer cell pathogenesis. Enzyme activity is undetectable in most normal cells and tissues, but present in immortal cells and cancer tissues. While expression of TERC, the RNA component of telomerase, is widespread, the restricted expression pattern of TERT, the telomerase catalytic subunit gene, is correlated with telomerase activity, and its ectopic expression in telomerase-negative cells is sufficient to reconstitute telomerase activity and extend cellular lifespan. We have used in situ hybridization to study TERT expression at the single-cell level in normal tissues and in various stages of tumour progression. In normal tissues, including some that are known to be telomerase-negative, TERT mRNA was present in specific subsets of cells thought to have long-term proliferative capacity. This included mitotically inactive breast lobular epithelium in addition to some actively regenerating cells such as the stratum basale of the skin. TERT expression appeared early during tumorigenesis in vivo, beginning with early pre-invasive changes in human breast and colon tissues and increasing gradually during progression, both in the amount of TERT mRNA present within individual cells and in the number of expressing cells within a neoplastic lesion. The physiological expression of TERT within normal epithelial cells that retain proliferative potential and its presence at the earliest stages of tumorigenesis have implications for the regulation of telomerase expression and for the identification of cells that may be targets for malignant transformation.
Nature Genetics 07/1998; 19(2):182-6. · 35.53 Impact Factor
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ABSTRACT: The expression of telomerase, the enzyme that synthesizes telomeric DNA de novo, is suppressed in normal somatic human cells but is reactivated during tumorigenesis. This reactivation appears to arrest the normal loss of telomeric DNA incurred as human cells divide. Since continual loss of telomeric DNA is predicted to eventually limit cell proliferation, activation of telomerase in cancer cells may represent an important step in the acquisition of the cell immortalization which occurs during tumor progression. The telomerase holoenzyme is composed of both RNA and protein subunits. In humans, mRNA expression of hTERT (hEST2), the candidate telomerase catalytic subunit gene, appears to parallel the levels of telomerase enzyme activity, suggesting that induction of hTERT is necessary and perhaps sufficient for expression of telomerase activity in tumor cells. To test this model directly, we ectopically expressed an epitope-tagged version of hTERT in telomerase-negative cells and show that telomerase activity was induced to levels comparable to those seen in immortal telomerase-positive cells and that the expressed hTERT protein was physically associated with the cellular telomerase activity. We conclude that synthesis of the hTERT telomerase subunit represents the rate-limiting determinant of telomerase activity in these cells and that this protein, once expressed, becomes part of the functional telomerase holoenzyme.
Oncogene 03/1998; 16(9):1217-22. · 6.37 Impact Factor
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M Meyerson, C M Counter,
E N Eaton,
L W Ellisen,
P Steiner,
S D Caddle,
L Ziaugra,
R L Beijersbergen,
M J Davidoff,
Q Liu,
S Bacchetti,
D A Haber,
R A Weinberg
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ABSTRACT: Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal human somatic cells but is reactivated during tumor progression. We report the cloning of a human gene, hEST2, that shares significant sequence similarity with the telomerase catalytic subunit genes of lower eukaryotes. hEST2 is expressed at high levels in primary tumors, cancer cell lines, and telomerase-positive tissues but is undetectable in telomerase-negative cell lines and differentiated telomerase-negative tissues. Moreover, the message is up-regulated concomitant with the activation of telomerase during the immortalization of cultured cells and down-regulated during in vitro cellular differentiation. Taken together, these observations suggest that the induction of hEST2 mRNA expression is required for the telomerase activation that occurs during cellular immortalization and tumor progression.
Cell 09/1997; 90(4):785-95. · 32.40 Impact Factor
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ABSTRACT: Telomerase is an RNA-directed DNA polymerase, composed of RNA and protein subunits, that replicates the telomere ends of linear eukaryotic chromosomes. Using a genetic strategy described here, we identify the product of the EST2 gene, Est2p, as a subunit of telomerase in the yeast Saccharomyces cerevisiae. Est2p is required for enzyme catalysis, as mutations in EST2 were found to result in the absence of telomerase activity. Immunochemical experiments show that Est2p is an integral subunit of the telomerase enzyme. Critical catalytic residues present in RNA-directed DNA polymerases are conserved in Est2p; mutation of one such residue abolishes telomerase activity, suggesting a direct catalytic role for Est2p.
Proceedings of the National Academy of Sciences 09/1997; 94(17):9202-7. · 9.68 Impact Factor
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C M Counter
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ABSTRACT: Telomeres cap and protect the ends of chromosomes from degradation and illegitimate recombination. The termini of a linear template cannot, however, be completely replicated by conventional DNA-dependent DNA polymerases, and thus in the absence of a mechanisms to counter this effect, telomeres of eukaryotic cells shorten every round of DNA replication. In humans and possibly other higher eukaryotes, telomere shortening may have been adopted to limit the life span of somatic cells. Human somatic cells have a finite proliferative capacity and enter a viable growth arrested state called senescence. Life span appears to be governed by cell division, not time. The regular loss of telomeric DNA could therefore serve as a mitotic clock in the senescence programme, counting cell divisions. In most eukaryotic organisms, however, telomere shortening can be countered by the de novo addition of telomeric repeats by the enzyme telomerase. Cells which are "immortal' such as the human germ line or tumour cell lines, established mouse cells, yeast and ciliates, all maintain a stable telomere length through the action of telomerase. Abolition of telomerase activity in such cells nevertheless results in telomere shortening, a process that eventually destabilizes the ends of chromosomes, leading to genomic instability and cell growth arrest or death. Therefore, loss of terminal DNA sequences may limit cell life span by two mechanisms: by acting as a mitotic clock and by denuding chromosomes of protective telomeric DNA necessary for cell viability.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 11/1996; 366(1):45-63. · 2.85 Impact Factor
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ABSTRACT: Telomeres are essential for function and stability of eukaryotic chromosomes. In the absence of telomerase, the enzyme that synthesizes telomeric DNA, telomeres shorten with cell division, a process thought to contribute to cell senescence and the proliferative crisis of transformed cells. We reported telomere stabilization concomitant with detection of telomerase activity in cells immortalized in vitro and in ovarian carcinoma cells, and suggested that telomerase is essential for unlimited cell proliferation. We have now examined the temporal pattern of telomerase expression in selected hematologic malignancies. We found that, unlike other somatic tissues, peripheral, cord blood, and bone marrow leukocytes from normal donors expressed low levels of telomerase activity. In leukocytes from chronic lymphocytic leukemia (CLL) patients, activity was lower than in controls in early disease, and comparable with controls in late disease. Relative to bone marrow, telomerase activity was enhanced in myelodysplastic syndrome (MDS) and more significantly so in acute myeloid leukemia (AML). Regardless of telomerase levels, telomeres shortened with progression of the diseases. Our results suggest that early CLL and MDS cells lack an efficient mechanism of telomere maintenance and that telomerase is activated late in the progression of these cancers, presumably when critical telomere loss generates selective pressure for cell immortality.
Blood 06/1995; 85(9):2315-20. · 9.90 Impact Factor
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ABSTRACT: We have measured telomere length and telomerase activity throughout the life span of clones of human B lymphocytes transformed by Epstein-Barr virus. Shortening of telomeres occurred at similar rates in all populations and persisted until chromosomes had little telomeric DNA remaining. At this stage, some of the clones entered a proliferative crisis and died. Only clones in which telomeres were stabilized, apparently by activation of telomerase, continued to proliferate indefinitely, i.e., became immortal. Since loss of telomeres impairs chromosome function, and may thus affect cell survival, we propose that telomerase activity is required for immortality. We have now detected this enzyme in a variety of immortal human cells transformed by different viruses, indicating that telomerase activation may be a common step in immortalization.
Journal of Virology 06/1994; 68(5):3410-4. · 5.40 Impact Factor
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ABSTRACT: Telomeres fulfill the dual function of protecting eukaryotic chromosomes from illegitimate recombination and degradation and may aid in chromosome attachment to the nuclear membrane. We have previously shown that telomerase, the enzyme which synthesizes telomeric DNA, is not detected in normal somatic cells and that telomeres shorten with replicative age. In cells immortalized in vitro, activation of telomerase apparently stabilizes telomere length, preventing a critical destabilization of chromosomes, and cell proliferation continues even when telomeres are short. In vivo, telomeres of most tumors are shorter than telomeres of control tissues, suggesting an analogous role for the enzyme. To assess the relevance of telomerase and telomere stability in the development and progression of tumors, we have measured enzyme activity and telomere length in metastatic cells of epithelial ovarian carcinoma. We report that extremely short telomeres are maintained in these cells and that tumor cells, but not isogenic nonmalignant cells, express telomerase. Our findings suggest that progression of malignancy is ultimately dependent upon activation of telomerase and that telomerase inhibitors may be effective antitumor drugs.
Proceedings of the National Academy of Sciences 05/1994; 91(8):2900-4. · 9.68 Impact Factor
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Cold Spring Harbor Symposia on Quantitative Biology 02/1994; 59:307-15.
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ABSTRACT: Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.
The EMBO Journal 06/1992; 11(5):1921-9. · 9.20 Impact Factor
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Experimental Gerontology 27(4):375-82. · 3.74 Impact Factor
