PERSPECTIVES
Regulators of Telomerase Activity

Laura J. Mauro and Douglas N. Foster

Department of Animal Science, University of Minnesota, St. Paul, Minnesota

	Telomeres and Telomerase

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The ends of eukaryotic chromosomes are capped by specialized structures called telomeres. These structures are comprised of between hundreds and thousands of tandem telomeric DNA repeats that are associated with several functional and regulatory proteins (Figure 1). The telomeres are believed to play an important role in the genomic stability of the cell, serving to protect chromosomes and allow for their complete end replication (1, 2). Normal cellular DNA polymerases fail to completely replicate the 3' end of the lagging strand of linear DNA molecules. As a result, as many as 200 nucleotides of telomeric sequence can be lost with each cell division without sacrificing functional genomic DNA. Recurrent loss of the telomere could eventually result in an uncapped or unprotected chromosome which is at greater risk for degradation, recombination, or fusion by cellular DNA repair mechanisms (3). If the cell senses DNA damage or single-stranded DNA ends, a decision is made to either repair the damage or begin the processes of cellular senescence and/or apoptosis.

View larger version (25K): [in this window] [in a new window]   Figure 1.   Telomeres are structural DNA elements which cap the ends of chromosomes. A metaphase chromosome is pictured where the end is enlarged to illustrate the linear structure of telomeric DNA. This DNA is usually positioned in a "t-loop" conformation as shown, with multiple proteins associated with the telomeric repeats, represented as ovals and circles in this diagram. As shown in the boxed inset, the telomere is composed of tandem repeats of the hexanucleotide sequence, TTAGGG, often as double-stranded DNA with a short single-stranded 3' overhang.

The critical shortening of the telomeres appears to function as a molecular clock. After a certain number of divisions accompanied by subsequent telomere loss, cells are no longer capable of dividing. This may signal normal cells to enter a state of replicative senescence (4). For example, these shortened telomeres may activate the expression of telomere shortening-sensitive genes (TSSG), such as the tumor suppressor p53, leading to cell growth arrest and apoptosis (5). Interestingly, all immortal cell lines examined to date appear to have no further loss of telomere length with each passing cell division, suggesting that telomere maintenance is a requirement if cells are to escape senescence and grow indefinitely (6). It is noteworthy that not all normal, immortal, or tumor cells re-express detectable levels of telomerase, so alternative telomerase-independent pathways for telomere length maintenance have been proposed (7, 8).

One approach to reset the molecular clock and restore the telomeric length is by the ectopic expression of the enzyme, telomerase. This enzyme is capable of synthesizing telomeric DNA de novo, thus capping the ends of telomeres leading to chromosomal stability (9). The telomerase reverse transcriptase (TERT) is a highly-conserved (10) ribonucleoprotein that utilizes the 3' telomeric end of the DNA as a primer and an RNA component as a template for the synthesis of G-rich telomeric repeats. Even though the primary structure of the RNA has evolved among species, there is a core secondary structure that is highly conserved among distant evolutionary groups (11). Furthermore, additional regulatory factors such as TRF1 and TRF2 (12), tankyrase (13), and TIN2 (14) have been shown to be physically associated with the telomeric end, maintaining a large tailed loop conformation termed the "t-loop" (see Figure 1). Telomeric t-loops have been isolated from humans and mouse (~ 3-18 kb in size) and from protozoa (~ 1 kb), suggesting that these structures are also evolutionarily conserved (15, 16).

	Regulation of Telomerase in Lung Tissue

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There is accumulating evidence that reversible telomerase regulation exists and is associated with cellular changes such as oncogenesis, differentiation, and cell injury. Differentiation of basal/stem cells in highly regenerative tissues such as the immune system, skin, and intestine, is often associated with repression or loss of telomerase activity (17). Human somatic cells often have undetectable telomerase activity, whereas telomerase is activated in at least 85% of cancerous tissues (4, 18). One of the first reports of telomerase activity in lung tissue indicated that all of the lung tumors and two of three virally-transformed lung cell lines analyzed were positive for telomerase activity (4). More recently, telomerase activity was demonstrated in 80% of 100 lung tumors analyzed, and the activity was associated with p53 overexpression and genetic instability (19). Telomerase activity was also found in non-small cell lung carcinomas and was shown to correlate with smoking status in 86% of the tissues sampled (20). Injury to lung tissue also can induce telomerase activity, as demonstrated in silica-instilled rat lung following fibroblast proliferation (21). A similar lung injury model in rats has been used by Nozaki and colleagues (22) to show that bleomycin-induced lung injury caused an increase in the number of rat lung fibroblasts along with pulmonary fibrosis and induced telomerase activity primarily in non-myofibroblast cells.

In this issue, Liu and colleagues extend the above studies by asking to what extent the cytokines, basic fibroblast growth factor (bFGF) and interleukin-4 (IL-4), act as potential mediators for regulating telomerase activity (23). They found that bFGF could induce telomerase activity in lung fibroblast cells from normal rats (NRF) and to even greater levels in lung fibroblast cells from bleomycin-treated rats (BRF). Treatment with bFGF was shown to cause a transient increase in both telomerase mRNA and protein expression. Conversely, it was shown that IL-4 inhibited telomerase induction in BRF cells, possibly as a result of their conversion to a myofibroblast phenotype. The authors point out that this is consistent with the preferential expression of telomerase activity in normal basal fibroblasts relative to differentiated myofibroblast cells.

	Mechanisms of Telomerase Regulation

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The activation or repression of telomerase activity observed during such cellular changes can be mediated, at least in part, by transcriptional regulation of gene expression. Cloning of the human telomerase (hTERT) gene has revealed a promoter with a relatively high GC content lacking both TATA and CAAT boxes (24, 25). In addition, there are two E-box elements and five Sp-1 transcription factor binding sites toward the more proximal portion of the promoter as well as estrogen response elements. Studies have shown that binding of c-Myc to the E-box can activate transcription of the hTERT gene and that this induction depends on Sp1 binding to its sites in the hTERT promoter (26, 27). Dimerization of the transcriptional repressor, Mad, with c-Myc can antagonize this activation, resulting in repression of transcription (28). Overexpression of the tumor suppressor, p53, can decrease the expression of endogenous hTERT mRNA as well as directly repress transcriptional activation of an hTERT promoter construct (29). Direct chemical modification of DNA by histone deacetylation can also repress human telomerase transcription (32). In addition, hormones such as estrogen and progesterone can function as activators of this gene (35), whereas retinoic acid acts as a repressor. These (26) and other (38) proposed regulators of hTERT transcription are listed in Table 1. A more extensive review of telomerase regulators has recently been published by Ducrest and colleagues (46).

Telomerase activity can also be controlled by interactions with proteins that are thought to be required for the assembly and disassembly of the telomerase holoenzyme. These proteins seem to affect the higher order structure of the telomere in terms of making it accessible or not to telomerase (47). The human telomerase-associated protein 1 (hTEP1) is thought to be a regulatory subunit of the telomerase complex, serving as a scaffold for recruitment of other necessary proteins (48). Co-immunoprecipitation studies have shown that the tumor suppressor, p53, can associate with hTEP1 and telomerase and inhibit in vitro telomerase activity (49). The protein, TRF1, and its interacting protein, tankyrase, also associate with telomerase, and overexpression of TRF1 can lead to telomere shortening (50). The exact mechanisms by which these and other proteins change telomerase activity remain to be determined.

Human telomerase and associated proteins may exist as phosphoproteins in the cell and this phosphorylation may serve as another post-translational mechanism to regulate telomerase activity. It appears that activation of signaling pathways that results in serine/threonine and/or tyrosine phosphorylation of the telomerase protein may function in this regulation. Telomerase protein immunoprecipitated from human breast cancer cells has been shown to be associated with the serine/threonine protein kinase C (PKC; 51). Activation of the PKC pathway in these cells via the phorbol ester, PMA, results in enhanced phosphorylation and enzymatic activity of this telomerase protein. Analysis of the amino acid sequence of human telomerase has revealed two putative phosphorylation sites for another serine/ threonine kinase known as Akt (protein kinase B). Activation of the Akt kinase or incubation with recombinant Akt results in enhanced telomerase activity in human melanoma cells (52). The fact that incubation with recombinant protein phosphatase 2A can inhibit nuclear telomerase activity in human breast cancer cells (53) serves as additional evidence that serine/threonine phosphorylation may be an important regulatory mechanism. This telomerase activity can be reactivated by endogenous kinases within these cell lysates or activated by okadaic acid, an inhibitor of serine/ threonine phosphatases. Tyrosine phosphorylation of the telomerase protein also appears to be relevant because the tyrosine kinase, c-Abl, can associate with and phosphorylate telomerase, resulting in inhibition of activity (54). Fibroblasts isolated from c-Abl^/ mice also exhibit telomere lengthening, suggesting that the absence of c-Abl releases a repression of telomerase activity. It is interesting to note that c-Abl kinase is capable of mediating signals in response to DNA damage, such as double-strand breaks, caused by exposure to ionizing radiation (55). The result of this c-Abl activation includes growth arrest and apoptosis. Ionizing radiation treatment of MCF cells can induce the phosphorylation of telomerase and this phosphorylation is blocked by the expression of a kinase inactive c-Abl protein (54). These studies are suggestive of a direct link between a genotoxic agent and its effect on telomerase activity via phosphorylation.

The extensive research on the regulation of telomerase activity has shown that modulation of this enzyme is critical for cell growth and survival. There is evidence that this regulation can be mediated by changes in gene transcription, protein-protein interactions, and phosphorylation. There is a growing list of putative regulatory mechanisms and specific regulatory molecules. Future research will need to identify the intra- or extracellular signals and their associated signaling pathways that can activate these mechanisms, ultimately changing telomerase activity and telomere structure. In addition, the connections between such signals and observed changes during oncogenesis, differentiation or cell injury will need to be clarified. These regulatory mechanisms will undoubtedly prove to be very complex as well as interesting and have important applications for the diagnosis and treatment of multiple cancers.

	Footnotes

Address correspondence to: Douglas N. Foster, Ph.D., University of Minnesota, 1988 Fitch Ave., St. Paul, MN 55108.

(Received in original form March 27, 2002).

Acknowledgments: The authors would like to thank Jennifer Adam for assistance with artwork.

	References

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