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© 2000 American Society for Clinical Oncology
Role of Telomerase in Normal and Cancer CellsFrom the Department of Adult Oncology, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, MA. Address reprint requests to Matthew Meyerson, MD, PhD, Department of Adult Oncology, Dana-Farber Cancer Institute, Mayer Building 430, 44 Binney St, Boston, MA 02115; email matthew_meyerson@ dfci.harvard.edu. ABSTRACT ABSTRACT: Shortening of the telomeric DNA at chromosome ends is postulated to limit the lifespan of human cells. In contrast, activation of telomerase, the enzyme that synthesizes telomeric DNA, is proposed to be an essential step in cancer cell immortalization and cancer progression. This review discusses the structure and function of telomeres and telomerase, the role of telomerase in cell immortalization, and the effects of telomerase inactivation on normal and cancer cells. Moreover, data on the experimental use of telomerase assays for cancer detection and diagnosis are reviewed. Finally, the review considers the evidence regarding whether telomerase inhibitors could be used to treat human cancers. THE GENETIC MATERIAL of eukaryotic cells is dispersed on multiple linear chromosomes, a total of 46 chromosomes in each human somatic cell, for example. One could imagine that the presence of these multiple independent chromosomes offers great selective advantages during evolution. Linear chromosomes permit increased rates of recombination and allow random chromosomal shuffling during sexual reproduction. Thus the dispersal of DNA on many linear chromosomes serves to increase genetic diversity in a population. THE END-REPLICATION PROBLEM Eukaryotic cells, however, must pay a price for these advantages. Linear chromosomes pose two problems for the cell: how to protect the free DNA ends from degradation and fusion, and how to replicate the extreme termini of the DNA. Exposed 5' and 3' termini of DNA are highly susceptible to nuclease degradation. In addition, such exposed DNA ends are highly recombinogenic and can ligate with other free DNA ends or can integrate into the heart of chromosomal DNA.1,2 Furthermore, conventional DNA-dependent DNA polymerases cannot completely replicate the ends of chromosomes.3-5 Synthesis of the so-called lagging strand of DNA depends on the use of small RNA primers (Fig 1). Even if the most terminal RNA primer should hybridize to the furthest point at the end of the chromosome, a statistically improbable situation, that primer would get degraded after DNA replication, leaving a gap. In this manner, the 5' end of the lagging strand would be shorter than the 3' end of the leading strand. With each cycle of DNA replication, more and more terminal DNA would be lost. Eventually, such DNA deletion would lead to the loss of cell viability.
TELOMERES AND TELOMERASE Telomeres, DNA-protein complexes that cap the chromosome ends, have been evolved by eukaryotic organisms to solve the problems of DNA end protection. Specialized telomere synthesis mechanisms, such as the telomerase enzyme, solve the problem of DNA end replication. In most organisms, telomeres are composed of repeats of short guanine-rich sequence motifs,6,7 together with sequence-specific DNA-binding proteins. In humans and other vertebrates, the telomeric repeat sequence is 5'-TTAGGG-3'.8,9 There are roughly 1,000 to 2,000 such repeats at the end of each human chromosome, or approximately 6,000 to 12,000 base pairs of telomeric DNA.8,10 The vast majority of these repeats are double-stranded, with several hundred base pairs of single-stranded extension of the G-rich strand at the extreme 3' end.11-14 Sequence-specific DNA-binding proteins attach to the telomeric DNA repeats. These proteins help to maintain telomere stability and to regulate telomere length. In human cells, the TRF1 and TRF2 proteins are known to specifically recognize the TTAGGG repeat sequence.15-17 TRF1 seems to regulate telomere length by inhibiting telomere elongation once telomeres reach a critical size.18 TRF2, in contrast, suppresses end-to-end fusions between chromosomes and serves to stabilize chromosome ends.19 It is likely that these proteins form the scaffold for a larger, multiprotein complex; one candidate component of such a complex, a poly adenosine diphosphate–ribose polymerase localized to the telomeres and binding directly to TRF1, has recently been identified.20 In most eukaryotic organisms, the DNA component of telomeres is synthesized by a specialized reverse transcriptase enzyme, telomerase. The catalytic core of telomerase is composed of an RNA subunit that serves as the template for telomere addition21-24 and a protein subunit that acts to catalyze the telomere synthesis reaction.25-27 In humans, the RNA subunit is known as hTR (human Telomerase RNA), whereas the protein subunit is known as hTERT (human Telomerase Reverse Transcriptase). The corresponding mouse subunits are known as mTR and mTERT.28-30 The human telomerase complex catalyzes the synthesis of new single-stranded GGTTAG repeats at the end of the chromosome.31 The enzyme is an RNA-dependent DNA polymerase, or reverse transcriptase, that carries its own RNA template for DNA synthesis. The human RNA subunit, hTR, contains one and five-sixths repeats of the template complementary to the telomere repeat, the sequence 5'-CUAACCCUAAC-3'.23 The RNA subunit is believed to bind to the terminal telomere DNA repeat, after which the catalytic subunit catalyzes the addition of deoxynucleotides to the 3' end of this repeat, and the RNA template then translocates to the newly synthesized 3' end of the telomere (Fig 2).21,31
The catalytic subunit of telomerase is conserved in yeast,25,26,32,33 protozoans,25,34,35 and mammals.27,29,30,33,36-38 Given that it synthesizes DNA from an RNA template, it is not surprising that this enzyme subunit shows significant conservation with reverse transcriptase enzymes from retroviruses such as the human immunodeficiency virus.25,26,39-41 Inactivation of catalytic residues, conserved with the reverse transcriptases, eliminates telomerase activity.25-27,38,42,43 The similarity of telomerase to reverse transcriptases may be useful in developing therapeutic telomerase inhibitors, especially because reverse transcriptase inhibitors such as azidothymidine are already known to be effective antiretroviral therapies.44 Various specific inhibitors of telomerase enzymes are now under laboratory investigation. TELOMERASE AND CELL IMMORTALIZATION When placed into culture, most normal human somatic cells have a limited lifespan. Human fibroblasts, for example, can divide an average of 40 to 50 generations before they stop dividing.45 This growth arrest is known as cellular senescence, and the limited number of cell divisions is called the Hayflick limit.46 In contrast, many cancer-derived cells are immortal in cell culture and therefore have a mechanism to evade the Hayflick limit. It remains controversial whether the cell culture immortality of many cancer cells is a fundamental property, required for tumorigenesis, or a by-product of other genetic changes. Given that human cells can divide only a limited number of times in culture, many researchers wondered whether there is a "clock" that measures cell divisions. The discovery of delayed cell death, many generations after the onset of telomere shortening, in micro-organisms with telomere maintenance defects22,47 raised the question as to whether telomere shortening could be the cell division clock in human cells. Several groups began to investigate the fate of telomeres in human aging and during the passage of human cell cultures. Human somatic cells were found to have shorter telomeres than sperm from the same individuals, which suggests that human telomeres shorten during development.10,48 Furthermore, telomere length in somatic cells, both fibroblasts and leukocytes, from older individuals was decreased on average compared with telomere length in somatic cells from younger people.48,49 Finally, telomeres were found to shorten during the passage of fibroblasts in culture49 and in cancers compared with adjacent normal tissues.48 The telomere shortening observed in human somatic cells led to the generation of the following hypothesis (Fig 3): telomerase is shut off in human somatic cells, limiting their lifespan, whereas it is activated in cells that become immortalized in culture.50,51 It seems that there are two points of lifespan limitation in cultured cells. Transformation with viral and/or cellular oncogenes extends the lifespan of human cells beyond the first growth arrest point, known as senescence, but these transformed cells eventually enter a phase known as crisis, where cells suffer chromosome aberrations and massive cell death.52-54 Rare immortal cell clones escape crisis and survive by telomerase activation, according to this model.
This model was validated by studies of telomere length and telomerase activity in mortal and immortal human cultured cells. Analysis of telomere lengths in human embryonic kidney cells transformed with simian virus 40 T antigen (and in human B lymphocytes transformed with Epstein-Barr virus) shows a steady decline until crisis, and then telomere maintenance in the survivors. Concordantly, the telomerase enzyme is inactive in the precrisis mortal cells and is activated in the immortalized cell clones.54,55 Cloning of the key telomerase subunits has clarified the mechanism for telomerase activation. Whereas the human telomerase RNA subunit is expressed in both telomerase-positive and telomerase-negative tissues,56,57 the expression of the human catalytic subunit gene, hTERT, seems to be rate-limiting for telomerase activity. The hTERT mRNA is expressed in immortal, telomerase-positive cell lines but not in mortal, telomerase-negative cells.33,36,37 Furthermore, when cells become immortalized by activating telomerase, they do so by inducing the expression of hTERT.36 Overexpression of hTERT in previously telomerase-negative cells generates an active telomerase enzyme in these cells,38,42,58-60 confirming that hTERT expression is a key step in regulating telomerase activity. Even more dramatically, ectopic expression of the telomerase catalytic subunit gene in certain telomerase-negative cells is able to extend the lifespan of these cells, confirming the model that telomerase can overcome the limits of human cell mortality, at least in some cases. hTERT expression leads to significant lifespan extension in strains of mortal fibroblasts and retinal epithelial cells, allowing them to bypass the senescence limitation to cell growth.58,60 hTERT-immortalized BJ foreskin fibroblasts have been found to proliferate for an additional 200 population doublings compared with control cell clones that can double for approximately 80 generations.61 There are no changes in these immortalized cell populations that indicate transformation to an oncogenic phenotype.61-63 Expression of the telomerase catalytic subunit gene also permits the survival of oncogenically transformed cells beyond the crisis limit to cell lifespan.64-66 Although telomerase expression is sufficient to immortalize some cell types, such as fibroblasts, directly, other cell types, such as keratinocytes, require the cotransfection of an oncogene for the inactivation of other growth suppressing pathways and thereby for direct immortalization.67 The use of telomerase-based cell immortalization may offer a variety of practical applications in medical research and potentially even in improving therapies.58 Immortalized cell populations will be useful in studying normal human cells that had been hitherto impossible to culture. Furthermore, if the effectiveness of treatments such as bone marrow transplantations and skin grafts ever becomes limited by long-term replicative failure (as opposed to the current situation in which survival after bone marrow transplantation is limited by problems such as leukemia relapse and graft-versus-host disease),68 telomerase-based extended lifespan cells could be useful to improve such treatments. The experiments described above show that telomerase activation can be sufficient for immortalization. Is it also necessary? Two types of experiments address this issue: inactivation of telomerase enzymes in otherwise immortal cells and analysis of telomerase activity in a wide variety of immortal cells. Taken together, the results argue that telomerase activation may be necessary for immortalization of some cells, or in some circumstances, but can be bypassed in others. Analysis of telomerase activity in immortal cells has led to the identification of two classes of cells. In one class, as described above, telomerase is activated. In the second class of cells, telomerase activity remains undetectable, and telomeres seem to be maintained by a telomerase-independent mechanism known as alternative lengthening of telomeres.69,70 The mechanism for alternative lengthening of telomeres in human cells is unknown but is believed to be a recombination-dependent process analogous to those that maintain telomeres in rare survivors of telomerase-deficient yeast.32,71,72 Primary human tumors as well as immortalized cell lines have likewise been observed to include a population of telomerase-negative tumors, presumably surviving by alternative lengthening of telomeres.69 The frequency and generality of the alternative lengthening of telomeres in human tumors have not been established. In summary, telomeres shorten with the passage of cells in culture, the majority of human cells that become immortalized acquire telomerase activity, and the enforced expression of telomerase activity leads to lifespan extension of human cells. This lifespan extension has some potential to achieve a variety of medical and pharmaceutical uses. TELOMERASE EXPRESSION IN NORMAL AND CANCEROUS HUMAN CELLS An initial survey of telomerase activity in a variety of normal and malignant cell types using a sensitive polymerase chain reaction–based method, known as the telomerase repeat amplification protocol (TRAP) assay, suggested that telomerase activity is present in almost all tumor samples but could not be detected in normal tissues, except for germ cells in the ovary and testis.73 More detailed analysis of a variety of cell and tissue samples confirms the high frequency of telomerase activity in tumor samples (see review in Shay and Bacchetti74 ) but shows that many regenerating normal tissues are also telomerase-positive. The first normal human tissues shown to be telomerase-positive were normal lymphocytes that had been stimulated to divide.75-77 Other normal human cell types that exhibit telomerase activity include intestinal epithelium,78 esophageal epithelium,79 cycling endometrium,80-82 basal keratinocytes,83,84 cervical epithelium,84 and hematopoietic stem cells.85 In some cases, human tissues that are telomerase-negative become telomerase-positive when growth is stimulated, as in the cases of lymphocytes and uroepithelial cells.86 Studies of human telomerase catalytic subunit expression in normal tissues are consistent with telomerase activity measurements. In situ hybridization analysis of hTERT expression reveals that it is expressed in a variety of normal cells and tissues with high proliferative capacity, including many different types of epithelial cells and hematopoietic precursors as well as spermatogonia.87 Immunohistochemical studies with anti-hTERT antibody likewise show high levels of expression in normal colonic crypt cells.88 The presence of telomerase activity in normal cells and tissues has important implications for the use of telomerase assays in cancer diagnosis and for the use of antitelomerase inhibitors in cancer treatment. However, the functional significance of this telomerase activity in human cells is still controversial. In addition, it remains unclear whether the widespread presence of telomerase activity in human cancers is due to selection for functional telomerase during cancer progression or to the expansion of a telomerase-positive stem-cell population.89 TELOMERASE AS A DIAGNOSTIC MARKER The use of telomerase as a tool for cancer diagnosis held early promise but may be limited by the frequent presence of telomerase activity in a variety of normal cells and tissues (described previously in Telomerase Expression in Normal and Cancerous Human Cells). The examples of breast, lung, and bladder cancer diagnosis are discussed here; many additional references for other tumor types, as well as different perspectives on the role of telomerase activity in cancer diagnosis, can be found in other reviews.74,90 The vast majority of breast cancers exhibit telomerase activity. In three independent studies, 79% to 95% of breast carcinoma samples were positive for telomerase activity, in contrast with 11% to 45% of fibroadenoma samples.91-93 Within the carcinoma samples, there was no correlation in any of the studies between axillary node metastases and telomerase activity or between estrogen or progesterone receptor status and telomerase activity. Thus, although telomerase activity is associated with carcinoma of the breast, it is not sufficiently specific for either diagnostic or prognostic purposes. In the case of lung cancer diagnosis, detection of telomerase activity was compared with the detection of p53 or K-ras mutations to analyze metastases of non–small-lung cancer to regional lymph nodes. Telomerase activity was found in 54% (26 of 48) of the lymph nodes in which cancer cells were histologically undetectable.94 This high false-positive rate is probably due to telomerase positivity of normal lymphocytes75-77 within the lymph nodes. A different application of telomerase assays for lung cancer detection has shown more promise. Telomerase activity was used to screen bronchial washings and was positive in 18 of 22 patients with primary or metastatic lung cancers, compared with only one of 19 patients without cancer. In contrast, cytology was suggestive or conclusive of cancer in 14 of 22 patients with lung cancers and 0 of 19 patients without cancer.95 Thus at least in this study, the sensitivity of telomerase assays for cancer detection is roughly comparable to that of cytology. For detection of bladder carcinoma in urine specimens, telomerase proved far more sensitive than cytology or other available screens. In three independent studies, the sensitivity of telomerase was 85%, 70%, and 62%, compared with 51%, 44%, and not done, for cytology.96-98 As seen above, however, the limitation of telomerase assays were the high false-positive rates owing to inflammatory cells; although telomerase was not detected in urine from healthy individuals, 34% of patients tested in one study had telomerase-positive urine, predominantly from patients with cystitis.96 In summary, although there is not yet a diagnostic indication where telomerase assays have shown a clear clinical benefit, there may be settings where telomerase assays could be useful—for example, as adjuncts to cytology. Tissues and fluids that do not exhibit confounding factors, such as inflammation, could be the best candidates. The use of telomerase assays in cancer diagnosis remains an area of active investigation. TELOMERASE AS A THERAPEUTIC TARGET IN HUMAN CANCER The activation of telomerase during cell immortalization and the restricted pattern of telomerase activity within the body have raised the possibility that telomerase inhibitors might be useful agents that could selectively kill cancer cells while sparing normal cells. Studies of telomerase overexpression and telomerase inactivation in mice and in human cells shed some light on these possibilities. As discussed above, overexpression of telomerase can immortalize certain otherwise mortal human cells. In at least some cell types, this immortalization is a contributory step in the acquisition of a tumorigenic phenotype. It has hitherto been impossible to generate tumorigenic human cells by oncogene transfection. Recently, however, the cotransfection of SV40 large antigen, mutant H-ras, and the hTERT gene has been shown to be capable of transforming both human fibroblasts and human epithelial cells into tumor cells.99 Although the use of hTERT for transformation in this manner has not been successful for all cell types and oncogenes tested,61 this result shows definitively that hTERT expression, and presumably the resultant telomerase activation, contribute to tumor development. What happens, then, when telomerase activity is disrupted in an immortal, telomerase-positive human cancer cell? When the telomerase RNA subunit is disrupted by antisense RNA expression, antisense expressing cell clones exhibit cell growth arrest and cell death, although there is not total ablation of telomerase activity.23,100 When the telomerase catalytic subunit is inactivated by the expression of dominant-negative mutants of hTERT, cellular telomerase activity is completely inhibited in cell clones, which then undergo telomere shortening, eventually leading to chromosome instability and cell death.101,102 Most strikingly, the cell clones that express dominant-negative hTERT protein are no longer able to form tumors when injected into immunodeficient mice.101 Recently, telomerase inhibition with oligonucleotide analogs has also been shown to lead to cell death,103 adding further promise to the concept of telomerase inhibition with small molecules. One of the major limitations of targeting telomerase in malignancies is that telomeres must shorten considerably before the cells become inviable. This could take many cell generations, perhaps more than would be required for the cancer to be lethal. One approach that might circumvent this problem is the use of telomerase as a tumor antigen. Peptides from the telomerase catalytic subunit hTERT were shown to elicit a cytotoxic T-lymphocyte response that could lyse hTERT-positive tumor cell lines.104 This approach has not yet been tested in vivo, and the effects on normal telomerase-positive cells within the body remain unknown. Nevertheless, such approaches merit further investigation. The above experiments show that telomerase can contribute to tumorigenesis and that telomerase inactivation can lead to delayed cell death, at least in some cell clones. The other important issue in telomerase inhibition is the possible effect of such intervention on normal cells within the body. This has been best analyzed in mice that lack the telomerase RNA subunit, mTR, and therefore lack functional telomerase. Mice that lack telomerase are viable and fertile, despite undergoing progressive telomere shortening.105 These mice can reproduce for six generations in the absence of telomerase, although the average telomere length gets shorter with each generation. In the sixth generation without telomerase, the mTR -/- mice become infertile and show cell proliferation failures in rapidly dividing tissues such as the hematopoietic system.106 The lifespan of sixth-generation mTR -/- mice is decreased in comparison with that of wild-type mice, and these mice show a number of degenerative and age-related defects, including increased rates of hair greying and alopecia, intestinal atrophy, delayed wound healing, and impaired stress responses.107 In summary, telomerase is required for the long-term viability and survival of mice, especially of proliferative tissues. These phenotypes are more accelerated when telomerase-deficient mice are bred into mouse strains with short telomeres.108 It is likely that humans, with an average telomere length of approximately 10 kb,8,10 would show more accelerated effects of losing telomerase activity than do Mus musculus, whose telomeres are approximately 40 kb long.109-111 Mouse cells without telomerase also show impaired survival in culture after a lag period. Although mouse embryo fibroblasts derived from the mTR -/- mice could proliferate for at least 200 generations,105 longer-term passage of mTR -/- mouse embryonic stem cells uncovered a markedly decreased growth rate after approximately 300 population doublings, followed by growth arrest after approximately 450 to 480 population doublings.112 It is not yet known whether telomerase-independent survivors can arise from these populations. The growth defects of the mTR -/- embryonic stem cells could be restored by ectopic expression of mTR.112 In summary, telomerase is essential both for long-term organismal viability and for long-term mitotic cell proliferation. Some of the more intriguing results from the telomerase knockout mice have related to the incidence of spontaneous cancers in these mice. Mice in the fourth to sixth generations without telomerase have a significantly higher cancer incidence than do wild-type mice, with four- to six-fold more tumors.107 These telomerase-negative tumors, mostly lymphomas and teratocarcinomas, originate predominantly from highly proliferative tissues. Consistent with this observation, deficiency of the p53 tumor suppressor gene seems to increase the viability of telomerase-deficient mice,113 suggesting that p53-deficient cells are likely to survive better without telomeres than do wild-type cells. The mechanism of the increased tumor incidence in telomerase-deficient mice is most likely to be an increase in chromosome instability and the resulting genomic alterations.48,107 In contrast, for mice that carry mutations in at least certain tumor suppressor pathways, such as the INK4a pathway, the incidence of tumors is significantly decreased in later generations of telomerase-deficient mice compared with mice with functional telomerase.114 These divergent effects of telomerase deficiency on tumor incidence in various mouse genetic backgrounds could depend on the presence or absence of intact DNA damage-repair responses, which could shut down cell growth in response to telomere dysfunction.114 In summary, there are cell types and pathways in which telomerase activation seems to promote tumorigenesis, but conversely there are other settings in which telomerase seems to protect cells from malignant transformation. Telomerase inactivation can kill cancer cells but is clearly toxic to proliferative normal cellular components as well. Because most effective cancer treatments vary significantly in their effects on different malignant cells and have varying toxicities for normal cells as well, the test of the role of telomerase inhibitors in human cancer will remain an empirical one. The experiments described above, despite the complex relationship between telomerase activity and tumorigenesis, provide a rationale for testing telomerase inhibitors when such molecules become available. The telomere hypothesis of cancer cell immortalization—telomere shortening limits the lifespan of telomerase-negative normal cells, whereas telomerase activation in cancer cells extends their lifespan—remains an attractive but not fully proven concept. Telomerase activation has been shown to extend the lifespans of otherwise mortal human cells in culture and can contribute to experimental tumorigenesis. 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