Multiple Levels of Telomerase Regulation
Telomeres are the nucleoprotein complexes found at the end of all linear chromosomes. These complexes consist of a six-base pair DNA repeat (TTAGGG)n and a growing list of associated proteins(1, 2) . Proper maintenance of these structures is required for ongoing cellular replication and genomic stability. Indeed, as normal cells divide in culture their telomeres progressively shorten, and upon reaching a critical length, cells cease to divide (3) . In contrast to what is observed in normal human cells, immortal tumor cells display stable telomere lengths despite continued cellular division(4, 5) . Overexpression of the catalytic component of telomerase (hTERT) in normal human cells leads, in many cases, to immortalization, demonstrating an important role for hTERT in cellular immortality(6, 7) . These observations form the basis of the telomere hypothesis that states that maintenance of telomere length is a prerequisite for cellular immortality (8) . Recent work by numerous groups has discovered that telomere maintenance is more complex than simply the maintenance of length, and it is clear that the overall structure of the telomere is also important for continued cellular proliferation (9) .
Expression of hTERT and the RNA template component hTR is sufficient for in vitro telomerase activity (10, 11) . Most human cells constitutively express hTR throughout development, whereas the expression of hTERT is more tightly regulated (5) . Early human embryonic cells express hTERT (12) ; however, hTERT expression and telomerase activity is not detectable in the majority of differentiated somatic cells (5) . Notable exceptions to this observation are tissue-specific stem cells and both activated B and T lymphocytes (13–16) . Although these cell types do express hTERT, the expression is not sufficient to maintain telomeres. In contrast to somatic cells, greater than 90% of human cancer cells utilize telomerase to maintain stable telomere lengths (5) . The remaining cancer cells utilize an alternative mechanism of telomere maintenance (ALT), which is defined operationally as telomerase-independent telomere maintenance and may consist of a recombination mechanism (17) . Ectopic expression of hTERT is sufficient to immortalize many normal human cells, indicating that hTERT activity is the rate-limiting component (6, 7) . A paradigm has emerged over the last few years whereby cancer cells utilize a number of approaches to activate the telomerase holoenzyme, and the recent report by Wong et al. adds yet another possible level of telomerase regulation (18) .
Because tumor cells express hTERT and have robust telomerase activity, the majority of studies to date have focused on the transcriptional regulation of hTERT in tumor cells. The hTERT gene is located on the distal arm of chromosome 5p (5p15.33) (19, 20) . Mutations in this region are rare, although amplifications have been detected in some types of cancers, suggesting that increased copy number may be one mechanism that increases telomerase expression in human tumors (19, 21) . The hTERT promoter contains a number of regulatory sites including two c-Myc binding sites (22) . The transcription factor c-Myc and its antagonist Mad (23, 24) affect the transcription of hTERT . Because MYC and MAD are sometimes perturbed in human cancers, this may provide another level of telomerase deregulation. [Estrogen also modulates hTERT transcription(25, 26) .] It has also been suggested that the hTERT promoter might contain a site that negatively regulates its expression in a p53-dependent manner (27) . Because the majority of human cancers are deficient in p53 protein or in functional p53-mediated pathways, this too could contribute to increased hTERT expression.
In a recent publication, Wong et al. demonstrated that subnuclear (nucleolar) shuttling of hTERT may also modulate telomerase activity at the telomere (18) . In the current study, this group created a green fluorescent protein–telomerase (GFP–hTERT) fusion protein and followed its fate throughout the cell cycle; ectopic expression of this fusion protein was capable of maintaining stable telomere lengths and immortalizing cells. They observed that in normal human fibroblasts this fusion protein was restricted to the nucleoli throughout much of the cell cycle; however, the protein relocalized to the nucleoplasm during late S/G2, the time at which human telomeres are thought to be replicated. Analyses of in vitro telomerase activity (TRAP) indicated that there was no change in enzymatic activity throughout the cell cycle. This suggests that the cellular localization of hTERT does not affect the enzymes’ ability to elongate telomeres but rather that the restricted localization of hTERT may sequester it from the 3’ termini of the chromosome, thus inhibiting telomere elongation at inappropriate times.
Unlike what was observed in normal cells, telomerase-positive transformed cells displayed a nuleoplasmic distribution throughout the cell cycle. In addition, when hTERT was overexpressed in ALT cell lines, it was also found in the nucleoplasm throughout the cell cycle. Similarly, introduction of the SV40 Large T antigen (LT) into normal human cells abrogated the nucleolar localization of hTERT, suggesting that it is the deregulation of the p53- and/or retinoblastoma-(Rb)-dependent pathways that leads to inappropriate localization of hTERT. This observation is interesting because it suggests that p53 may influence telomerase activity at two distinct levels. At one level, loss of p53 may contribute to increased transcription of hTERT (27) . This study suggests that p53 expression may also control telomerase activity at a second level by altering the subcellular localization of the holoenzyme. The potential role of the Rb pathway in this regulation remains to be determined. Together, these observations suggest that telomerase activity is sequestered in the nucleoli in cells with intact cell cycle checkpoints. Upon inactivation of these pathways, hTERT is released from the nucleoli, presumably giving telomerase access to the telomeres thoroughout the cell cycle, resulting in inappropriate elongation and maintenance of the telomere.
Maturation of the telomerase complex requires nucleolar components (28) ; therefore, it is not necessarily surprising to find hTERT in the nucleolus. However, the fact that hTERT is restricted to the nucleolus during particular phases of the cell cycle suggests that there is an important consequence to hTERT localization. To demonstrate whether this observed nucleolar localization was of functional importance, Wong and colleagues exposed cells expressing GFP–hTERT to γ -radiation. They found that when normal cells expressing GFP-hTERT were exposed to γ -radiation, nucleolar localization was enhanced. Surprisingly, they also found that in normal cells expressing LT, in transformed cells, and in cells that utilize ALT for telomere maintenance, GFP-hTERT also relocalized to the nucleolus following γ -irradiation. In in vitro assays, the association of telomerase with DNA is promiscuous, and it has been suggested that telomerase can participate in chromosome healing following double-strand breaks (29) . Addition of telomeric repeats to internal sites in the chromosome would likely have disastrous effects on the genome. Therefore, one possible explanation for this observation is that sequestration of the telomerase complex in the nucleolus might not only be important for limiting hTERT’s access to the telomere at inappropriate times throughout the cell cycle, but also during times of DNA damage. In addition, because this DNA damage–induced relocalization of hTERT occurs in both normal cells and cells expressing LT, it indicates that there is a mechanism that is distinct from the cell cycle control of nucleolar localization of hTERT, which is p53 and/or Rb independent. Understanding this mechanism will require further investigation but highlights the complexity of hTERT regulation.
Telomere homeostasis plays an important role in both normal and malignant cell physiology. Controlling the expression and activity of the telomerase holoenzyme is complex, and the current study (18) unravels another layer of this regulation. A number of other questions arise, including what mechanisms control the subcellular localization of hTERT and how these mechanisms are perturbed in human cancers. Because of its restricted expression, telomerase is an attractive target for future antineoplastic approaches, and the study by Wong et al. (18) provides yet another level at which future therapies can be directed.
- © American Society for Pharmacology and Experimental Theraputics 2002
References
Sheila A. Stewart, PhD, is a postdoctoral fellow at the Whitehead Institute for Biomedical Research. E-mail sstewart{at}wi.mit.edu
