Extension of Life-Span by Introduction of Telomerase into Normal Human Cells

Abstract

Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

Expression of mouse telomerase reverse transcriptase during development, differentiation and proliferation

“…Expression of the hTERT gene and concomitant activation of telomerase has been demonstrated to be su cient for extension of cellular replicative lifespan and bypass of the Hay¯ick limit for three di erent primary human cell lines (Bodnar et al, 1998). If the regulation of TERT expression indeed represents a key gatekeeper for the transition to an immortal phenotype in the mouse, as it appears to be for human cells, then the continued expression of TERT in mouse cells and tissues could be an important permissive factor enabling their cancer-prone phenotype.…”

“…Most somatic human tissues and primary cells possess low or undetectable telomerase activity, leading to a steady decline in telomere length with continued organ renewal in vivo and with passage in culture. This progressive telomere shortening has been shown to provide a signal for entry of cells into a state of replicative senescence (Harley et al, 1990;Bodnar et al, 1998;Kim et al, 1994).…”

Regulation of telomerase activity by the p53 family member p73

“…Although some tumor cells maintain telomere length by the alternative lengthening of telomere (ALT) pathway involving recombination processes, >85% of all tumor cells rather do so by inducing telomerase activity through hTERT transcriptional upregulation . Indeed, ectopic expression of hTERT in telomerasenegative cells is sufficient to restore telomerase activity and induce the immortalization of several primary human cell types (Bodnar et al, 1998). However, although hTERT expression alone is sufficient to immortalize for example human fibroblasts in culture, spontaneous immortalization is extremely rare.…”

Induction of telomerase activity in v-myc-transformed avian cells

“…A signal generated by telomeres shortened below a threshold value would induce cells, which have passed through a number of genetically determined cell cycles, to reach a quiescent state de®ned as replicative senescence (Harley, 1991). This hypothesis has recently received indirect support from the demonstration that the forced expression of the catalytic subunit of telomerase in normal human cells is su cient to prevent replicative senescence (Bodnar et al, 1998;Vaziri and Benchimol, 1998). Although there are instances in which human and murine cells are capable of unlimited cell doublings in the absence of active telomerase Blasco et al, 1997), telomerase reactivation could represent a crucial advantage for the unrestrained growth of tumor cells.…”

“…Telomerase activity was measured by the telomere repeat ampli®cation protocol (TRAP) (Kim et al, 1994), as modi®ed in Falchetti et al (1998). As shown in Figure Alterations in telomere length have been shown in human cells as consequence of the forced expression of telomerase catalytic subunit (Bodnar et al, 1998). We have used terminal restriction fragment (TRF) length assay (Strahl and Blackburn, 1966) to measure average telomeric lengths and have found no di erence between early passage (P1, equivalent to 3 PD in vitro) chicken NR cells and late passage (P15, equivalent to about 40 PD) cells transformed by either v-Myc or v-Src (not shown).…”