Telomeres, Telomerase, and their Role in Oncogenesis

Cassidy Crook

A Historical Perspective
Eukaryotic chromosome-ends are equipped with specialized protective caps known as telomeres. Telomeres were first described by Herman Muller in the early 1930s following a series of experiments using x-rays to generate artificial Drosophila mutants by fragmenting chromosomes. Muller found the chromosome terminal-region to be resistant to such fragmentation and thus concluded the terminal gene must have a special function, that of sealing the end of the chromosome, so to speak, and that for some reason a chromosome cannot persist indefinitely without having its ends thus sealed (Muller, 1938; Mcknight & Shippin, 2004). In the late 1930s Barbara McClintock observed similar unique qualities of chromosome ends while observing the dicentric chromosome breakage-fusion-bridge cycle in maize. McClintock established a system in which a dicentric chromosome 9 containing dominant alleles for plant color and seed characteristics in the bridge region was combined with a wild-type chromosome 9 with recessive alleles for the genes of interest. During anaphase/telophase the centromeres of chromosome 9 were directed to opposite spindle poles resulting in chromosome breakage at the bridge region. Generally, this cycle was completed by fusion of the broken sister chromatids to regenerate the divalent chromosome. However, somewhat inexplicably, McClintock discovered that the recessive alleles were consistently expressed when the system was introduced into embryonic tissues. She concluded that a mechanism of chromosome healing must be present to repair the broken chromosome ends in the embryonic tissue (McClintock, 1941; Mcknight & Shippin, 2004). Unknowingly, McClintock had stumbled upon the primary activity of what is now a much-celebrated enzyme, telomerase. A precise role for the phenomenon of chromosome healing was first eluded to in the early 1970s from the independent musings of Alexey Olovnikov and James Watson. Elucidation of the mechanism by which semiconservative replication is carried out led Watson and Olovnikov to intuitively realize that reverse elongation of the lagging strand through ligation of a series of Okazaki fragments would inevitably result in an unreplicated segment of DNA at the chromosome end. Should the RNA primer of an Okazaki fragment happen to align precisely with the chromosome end, then an unreplicated segment the length of the primer (~10-12 bp) would result; however, an unreplicated segment of somewhat greater length would result in all other instances. It is estimated the a chromosome loses approximately 50 to 200 bp per mitotic cycle (Kim et al. 1994). Thus, the apparent consequence of the end replication problem is continual shortening of the chromosome. Demonstrating remarkable insight, Olovnikov suggested that chromosome shortening may be linked to cellular senescence (Olovnikov, 1971, 1973; Watson, 1972). However, despite initial conjecture regarding telomeres and their function(s) our current understanding of chromosome termini has been most significantly influenced by Elizabeth Blackburn. Blackburn discovered the hexanucleotide sequence CCCCAA (i.e. TTGGGG on the nonsense strand) repeated in tandem while studying the termini of linear rDNA mini-chromosomes inTetrahymena thermophila (Blackburn & Gall, 1978). Following elucidation of the telomeric repeat sequence in Tetrahymena, similar telomeric repeats were reported for various other organisms. Humans were found to contain the sequence TTAGGG while the model plant Arabidopsis was determined to contain the sequence TTTAGGG, demonstrating a high degree of sequence conservation among species (Mcknight & Shippin, 2004). Thereafter, Carol Greider, working with Elizabeth Blackburn, demonstrated that when a 4-repeat TTGGGG oligonucleotide primer was added to Tetrahymena extracts the sequence was elongated with a six base pair periodicity. These findings led to the accumulation of sufficient evidence to conclude the presence of a telomere-synthesizing enzyme and, subsequently, the purification of the enzyme telomerase (Blackburn et al 2006).

Structure and Function Overview

Telomerase (TERT) is a ribonucleoprotein complex exhibiting de novo reverse transcriptase (RT) activity by incorporating a long RNA moiety (TR) containing a telomeric primer sequence. Telomerase is able to recognize and elongate the chromosome-terminal 3' overhang segment such that the complimentary strand may be filled-in by DNA polymerase through general lagging strand synthesis (Chan & Blackburn, 2003). TERT exhibits considerable structural homology to other RTs, containing seven characteristic RT motifs at its core: 1, 2, A, B', C, D, and E. Like many polymerase enzymes, TERT exhibits a general right hand form, distinguished by a palm-like region, fingers, and a structure resembling a thumb. However, several TERTspecific sequences have been identified, including a large insertion between the conserved A and B' motifs, constituting an augmentation of what would be the fingers domain; this insertion has been coined IFD for inter-fingers domain. In addition, TERTs often exhibit an N-terminal extension (NTE) as well as a Cterminal extension (CTE) and stabilization of the ribonucleoprotein heterocomplex is dependent on a crucial RNA-binding domain (TRBD) (Autexier & Lue, 2006). The telomere transferase activity of TERT is thought to be primarily directed by the reverse transcriptase domain, TRBD, and the CTE region. The RT domain encompasses the palm and fingers subdomains and contains the enzymes active site. Crystallographic studies have indicated that the active site is characterized by an aspartic acid triad in the palm domain at the A and C motifs; this acidic cluster likely acts to chelate two metal cofactors required for nucleotide incorporation. The CTE region exhibits no evolutionary sequence homology; however, its spatial orientation suggests that it is akin to the canonical polymerase thumb domain. When in association with TRBD, the CTE participates in the formation of a central ring-like structure that appears to accommodate the RNA-DNA heteroduplex (Gillis et al. 2008).

Senescence and Immortality

In 1961 Leonard Hayflick and Paul Moorehead conducted a series of experiments wherein the replicative capacity of human male fibroblasts at the fortieth doubling was compared to female fibroblasts at the tenth doubling by comparing cultures for the individual cell types with cultures of male and female cells mixed in equal amounts. When the control population of male cells ceased to divide the mixed culture was examined and found to contain only female fibroblasts (Hayflick & Moorehead, 1961). Based on these results, Hayflick described what he called the Phase III phenomenon, in which cultured cells reach a replicative limit and can no longer divide; this phenomenon is today known as the Hayflick limit. Hayflicks limit challenged the widely accepted idea that cells in culture could divide indefinitely and were essentially immortal. Moreover, Hayflick suggested that this phenomenon of cellular senescence was governed by an intrinsic celldivision counting mechanism, a sort of molecular clock, described as a replicometer (Hayflick, 1998). Importantly, Hayflick proposed a relationship between cellular senescence and aging and, in addition, recognized that the replicative counting mechanism must somehow be bypassed in order to induce a tumorigenic phenotype. Today it is accepted that the phenomenon of telomere shortening in the absence of telomerase is the mechanism by which cellular senescence is manifest. Critical shortening of telomeres past a threshold length results in crisis (i.e. DNA damage-response induced apoptosis), replicative arrest, or proliferation accompanied by widespread genome instability (Dahse et al. 1997). Interestingly, most somatic cells repress telomerase expression while rapidly dividing tissues, including testies, ovaries, and hematopoietic cells, as well as stem cells and some oncogenic transformants, have been shown to exhibit telomerase activity (Autexier & Lue, 2006). Furthermore, myriad studies have demonstrated that TERT-deficient cells transformed to express the enzyme may subsequently become immortal (Counter et al. 1992; Dahse et al. 1997; Bodner et al 1998).

Telomerase and Cancer

The first striking evidence for an inherent relationship between telomerase, cellular immortalization, and carcinogenesis was the observation that HeLa cells, infamous for their highly proliferative, malignant, and immortal phenotype, constitutively express telomerase (Morin, 1989). Several years later it was found that 90 of 101 tumor lines representing 12 cancer types exhibited telomerase activity, while no telomerase activity was detected in 50 samples of somatic tissue (Harley et al. 1994). Telomerase activation represents a critical point in the development of the cancer phenotype, yet, telomerase activity is not sufficient to promote neoplastic progression. Rather, the process of oncogenesis is thought to be initiated solely through the silencing of tumor suppressor genes and the up-regulation of oncogenes. Notably, Calvin Harley (2002) argues that telomerase is not an oncogene because it doesnt fit the archetypal perception of an oncogene. Unlike other oncogenes, telomerase is not expressed as a protooncogene and its role in carcinogenesis is not exclusively associated with disorder or deregulation of the cellular machinery. Despite this, the fundamental nature of cancer as a disease characterized by a high frequency of unregulated cell proliferation is wholly dependent on the involvement of a mechanism by which the end replication problem and replicative senescence may be evaded (Wai, 2004). Strangely, stable yet extensively shortened telomeres have been observed repeatedly in tumor cells. Experimental observations from the artificial induction of immortality in human fibroblasts by transformation with telomerase suggest that shortened telomeres in cancer cells are an artifact of the delayed activation of telomerase during oncogenesis. It has been proposed that the disregulation of cell cycle checkpoints during carcinogenesis permits cells to exceed their replicative capacity such that telomeres are shortened to a hyper-critical length, switching on telomerase expression (Wai, 2004). Thus, for a cancer cell the activation of telomerase essentially represents a sort of cell rescue event which simultaneously enables replication to proceed ad infinitum. The processes of cell immortalization by telomerase and carcinogenesis are too often portrayed as distinct phenomena rather than being viewed as convergent cooperative and reciprocal processes; while telomerase does not itself possess tumor suppressor or oncogenic properties, its role in the early stages of transformation may be more influential than the general perception allows. It has been demonstrated that immortalized cells are more readily transformed into tumor cells; in particular, tumorigenic transformation of wild-type hamster fibroblasts by the oncogene ras has been shown to be ineffectual, while transformation of immortalized fibroblasts successfully results in tumorigenesis (Hahn & Weinberg, 2002). Perhaps such findings warrant a reassessment of the idea that oncogenes and tumor suppressor genes are solely responsible for driving the early stages of carcinogenesis.

Telomerase as a Therapeutic Target

Ideally, a therapeutic target for cancer is that which is expressed solely in the cancerous tissue and is critical to the maintenance and survival of the malignant phenotype. Our current understanding of telomerase is sufficient to consider it as a therapeutic target for cancer. Not only is telomerase critical to the survival of cancer cells, but it is expressed in few somatic tissues. That said, telomerase is expressed in reproductive tissues, juvenile hematopoietic cells, and stem cells thus, presenting a significant, though perhaps manageable, challenge for therapeutic approaches. Transformation of human tumors with dominant-negative hTERT has demonstrated that loss of telomerase function arrests tumor growth (Kelland, 2005). Several methods have been considered for inhibiting telomerase function in humans. The design of synthetic inhibitory drugs is perhaps the most evident approach to targeting telomerase. However, it has additionally been proposed that a mutant TR strand could be introduced into tumor cells to effectively reduce telomerase activity (Wai, 2004). Still, not all tumors utilize telomerase to maintain telomere length. A secondary

mechanism known as alternative lengthening of telomeres (ALT) has been described. In a general sense, ALT utilizes a recombinatorial process to transfer long terminal segments, resulting in highly heterogeneous telomere lengths. Notably, ALT could potentially be exploited by tumor cells as a mechanism for resistance to therapeutic approaches targeting telomerase (Kelland, 2005).

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