Telomerase activity is sufficient to allow transformed cells to escape from crisis.

Primary human and other mammalian cells, when cultured in vitro, generally exhibit a strictly limited proliferative life span, after which they permanently exit the cell cycle but remain viable for long periods in a state termed senescence (12, 53). The predominant theory of senescence is that growth arrest is a response to telomere shortening, which occurs as a result of incomplete replication of the extreme 3′ ends of linear chromosomes (29, 51). Telomerase, a multisubunit riboprotein, repairs and maintains telomeric ends in mammalian germ cells, in simple organisms such as yeast and tetrahymena (4), and in most tumor-derived cell lines (25, 40). The vast majority of normal, cycling somatic cells have no detectable telomerase activity (25). It is hypothesized that senescence occurs when the telomere lengths of one or more chromosomes reach a critical point at which cells are signaled to exit the cell cycle. This model was strengthened by the demonstration that the introduction of telomerase activity into normal, cycling (presenescent) primary cells prolongs the proliferative life span, perhaps indefinitely (6, 45). The normal limitation on life span can be bypassed by a variety of transforming mechanisms that disable key cell cycle regulators (7, 20, 53–55). Transformation by oncogenes such as simian virus 40 large T antigen (SVLT), human papilloma virus E6 and E7, and adenovirus E1A and E1B triggers entry into the cell cycle and is sufficient to delay senescence but fails to induce immortality in human cells (2, 18, 37, 42). Unchecked proliferation ultimately gives way to slowed growth, altered cell morphology, and high rates of cell death (21). This end point is most commonly referred to as “crisis,” although other terms, such as mortality checkpoint 2 or M2, have been used. Crisis and senescence share the property of growth arrest following a defined number of cell divisions. However, the well-controlled exit from the cell cycle, the preservation of chromosomal integrity, and the gradual loss of viability observed during senescence contrast with the proliferative slowing, chromosomal instability, and ongoing cell death that characterize cells in crisis. It is not known whether crisis and senescence share the same underlying etiology. Because crisis is characterized by severe chromosomal structural instability (33, 43) and because it has been demonstrated that transformation does not halt telomere shortening during replication (15, 16), it has been speculated that crisis occurs when telomere erosion has progressed beyond a second critical point such that chromosomes become unstable. The best evidence in favor of a relationship between telomere shortening and crisis is the high degree of correlation between immortalization and telomere stabilization. Depending on the cell type, 1 in 105 to 1 in 107 cells will escape crisis and go on to form immortal lines (24, 41). The rare clones that do emerge from crisis invariably exhibit some form of telomere stabilization, usually by telomerase (25) but sometimes by alternate mechanisms (9), implying that telomere stabilization is crucial to continued growth. Conversely, immortal HeLa cells can be forced into crisis when the telomerase holoenzyme is disabled by the expression of antisense hTR (the RNA component of the enzyme) (19). Specific chromosomal changes observed in crisis are consistent with telomere loss. Mean telomeric TTAGGG lengths reported for cells in crisis are so low (1.5 to 2 kb) (15, 16, 27, 29) as to raise the possibility that at least some chromosome ends may have been eroded into subtelomeric regions (1, 17, 31, 51). Intertelomere associations become common, and dicentric chromosomes increase in frequency (16, 21, 33, 37, 43). These are predicted consequences of telomere loss and are similar to chromosomal defects seen in serially passaged cells from mTR knockout mice (5). Despite the strong correlation between crisis and telomere loss (or immortalization and telomere stabilization), a causal relationship between these events has not been definitively established. As part of an effort to understand the relationship between pancreatic endocrine cell growth and differentiation, we have been developing cell lines from the human fetal and adult pancreas using a retroviral vector expressing SVLT and H-rasval12 (48, 49). The TRM-6 (49) and βlox5 cell lines were established from human fetal and adult pancreatic islets, respectively. Like other cells transformed or cotransformed with SVLT, these cells exhibit an extended life span but eventually enter a crisis phase characterized by increased cell death, altered morphology, and lack of proliferation. Although many human cell types expressing SVLT have eventually produced rare clones that escaped crisis and went on to form immortal lines, attempts to derive immortal clones from TRM-6 or βlox5 cells have been unsuccessful so far. Because telomerase activation may be the primary means by which cells escape crisis and because introduction of the human telomerase reverse transcriptase (hTRT) subunit (34) has been shown to reconstitute telomerase activity in several human cell types (35, 50), we tested the hypothesis that hTRT expression could rescue TRM-6 and βlox5 cells from crisis.