Crisis intervention: the role of telomerase.

Among the most mysterious problems from both a molecular biology and medical point of view are the steps leading to the transformation of differentiated quiescent cells into oncogenic cells, which are capable of continual replication and growth. Two studies, one by Counter et al. (1) published in a recent issue of the Proceedings and a second published in this issue of the Proceedings (2), provide further evidence for a direct role(s) of telomerase in oncogenic transformation. To investigate this process, the authors took advantage of a commonly used in vitro model system for defining steps in aging and in cellular immortalization in vitro (3). As first described by Hayflick (3), normal cultured cells proliferate until they reach a discrete point in which population growth ceases. This period is termed the M1 stage of aging, or replicative senescence (Fig. 1 red; ref. 3). This block, however, can be overcome by viral oncogenes. When cells are transformed with viruses that block p53 and pRB function (e.g., SV40), they continue to proliferate for an extended period of time (Fig. 1, blue). Ultimately, the cells reach a ‘‘crisis’’ point (M2) with a concomitant increase in both rates of death and chromosomal abnormalities (Fig. 1, blue; refs. 4–6). Only 1 3 1027 cells survive this stringent selection. Both M1 and M2 are therefore potential suppression pathways for tumorigenesis. In the context of this commentary, M1 and senescence and M2 and crisis are used interchangeably. It is the nature of the events in M2 (specifically the role of telomerase and telomeres) that is approached in these two reports. Telomeres, the termini of eukaryotic chromosomes, have a special problem in completing replication, termed the ‘‘endreplication’’ problem (7–9), which was proposed originally by Howard Cooke to be linked to the behavior of cells in M1 and M2 (10). This problem occurs as the consequence of the inability for the normal DNA replication system to overcome loss of terminal RNA primers (7, 8) and multiple exonucleolytic processes. In most eukaryotes, the enzyme that compensates for this loss is telomerase (9). Telomerase is a unique enzyme that adds simple sequence repeats (e.g., TTAGGG in humans) onto preexisting 39 overhangs. The core enzyme actually consists of two components: an RNA, which serves as template for the simple sequence tracts, and a catalytic subunit that acts as a reverse transcriptase on the RNA template (9, 11–13). Cloning and characterization of the human telomerase RNA (14) and the human catalytic subunit of telomerase (hTERT) (15, 16) have demonstrated that in many cell lines, hTERT, but not the telomerase RNA, is expressed concurrently with telomerase (17–19). These data suggest that hTERT, at least in some contexts, may be the limiting factor for telomerase activity. So, what is the evidence of a relationship between telomere length, telomerase, and the M1 and M2 mortality stages? One of the primary lines of evidence was provided by the observation that telomeres of cultured somatic cells continuously erode until M1 (6). Ectopic expression of hTERT leads to cellular immortalization and a bypass of senescence (20–22), suggesting that telomere size may be causally related to senescence. If M1 is overcome by transformation with viral oncogenes, telomeres continue to decrease in size until M2 is reached, a process that may be dictated by telomere length itself. Numerous additional studies have indicated that telomerase is involved in this in vitro system. First, telomerase activity and hTERT are absent in most primary tissue systems, while both are present in high abundance in immortalized cells (6, 15, 16). Second, whereas telomere size continually decreases during the aging process in vitro, immortalized cells reach an equilibrium, albeit at shorter-than-wild-type length. Third, continued expression of telomerase is necessary to maintain immortality (23). Finally, a loss of telomerase results in telomere shortening and subsequent inviability in yeast (24), similar to the observations in human cells. However, the role of telomerase and hTERT as cells enter crisis has not yet been investigated, until the studies from Counter et al. (1) and Zhu et al. (2). This is a central question because ectopic expression of hTERT is likely to best mimic the alterations that occur in the survivors of crisis. The immortalizing events in crisis may in turn be a model for the steps involved in the malignant pathway in vivo. By using a cultured HEK (human embryonic kidney) system (25), Counter et al. (1) demonstrated that cells that were virally transformed to overcome M1 and subsequently transfected with a vector containing hTERT became immortalized. Their studies indicate that telomeric tracts stabilize at sizes greater than those normally obtained during crisis. Counter et al. (1) therefore concluded that an increase in telomere size beyond an M2 telomere threshold, rather than telomerase activity per se, is responsible for immortalization (Fig. 1, yellow). In contrast, a C-terminal hemagluttinin-tagged hTERT derivative, although catalytically active (14), did not avert crisis, raising the possibility that a noncatalytic domain of hTERT may be involved in the immortalization process. However, it cannot yet be excluded that the abundance of telomerase contributes to the behavior of the tagged and untagged forms of hTERT. Zhu et al. (2) took an analogous approach to express the hTERT in cell lines derived from primary fibroblasts. Although this study confirmed the Counter et al. (1) investigation, the report also introduces some additional puzzles. Zhu et al. (2) provided evidence for two steps after immortalization of M2 cells: (i) continual loss of telomeric sequences below known crisis thresholds, followed by (ii) the stabilization of the telomeres at a size well below the M2 threshold (Fig. 1, yellow). They postulate, therefore, that telomere size is not the deciding factor for entering crisis. Rather, an additional, previously undescribed property of telomerase may be responsible for circumventing the consequences of short telomere sizes. Despite the discrepancies between the results shown in these two papers, a particularly intriguing result was demonstrated in both studies: transfection of the human catalytic subunit leads to immortalization of M2 cells, with the concurrent loss of