Telomeres are DNA-protein complexes at the ends of eukaryotic chromosomes essential for chromosomal integrity and normal cell growth (McEachern et al., 2000
; Blasco, 2002
). Vertebrate telomere DNA is composed of tandem repeats of the sequence TTAGGG and a 3′-overhang that forms a t-loop with the double-stranded DNA protecting the 3′ end of telomeres. Because conventional DNA polymerases cannot replicate the lagging strand at the 5′ end, telomeric DNA is progressively lost with each round of cell division, 50–150 base pairs per cell division in human cells, unless the cells express the ribonucleoprotein complex telomerase. The enzymatic core of telomerase consists of the reverse transcriptase TERT (te
ranscriptase) and its template RNA TR (t
NA). While TR is ubiquitously expressed, TERT expression is limited to germ cells and stem cells in the normal human body. When telomeres become shorter than the critical threshold in somatic cells due to a lack of TERT, p53- and Rb-regulated DNA damage responses trigger growth arrest (replicative senescence) (Maser and DePinho, 2002
). If TP53 and RB are inactivated, these cells can bypass this growth arrest, but the cells will eventually die because of massive chromosome end fusions triggered by the cumulative telomere loss (crisis).
The telomerase activity is subjected to multiple levels of regulatory mechanisms (Blasco, 2002
; Kyo and Inoue, 2002
). Transcriptional regulation of TERT by c-Myc, Max and Sp1 is one of the most critical control mechanisms. Alternative splicing of TERT also regulates the telomerase activity by producing more than six forms of transcripts including truncated forms and dominant-negative forms of TERT. Subcellular localization of TERT adds another layer of regulation to the telomerase activity. For instance, telomerase activity in activated T cells is not dependent on the total TERT protein level, rather the activity is defined by nuclear translocation of TERT accompanied by phosphorylation (Liu et al., 2001
). While transfected TERT is localized in the nucleoli in normal cells and released into nucleoplasm only in the S phase, it is always widely distributed in the cancer cell nucleoplasm (Wong et al., 2002
). In addition to these direct modifications of TERT, a number of telomere binding proteins play essential roles in modulating the telomere length (Blasco, 2002
). For example, TRF1 and TRF2 bind to the TTAGGG repeats and negatively regulate the telomere length through interactions with other proteins on telomeric DNA (de Lange, 2002
Since telomere length is tightly linked with cellular senescence, it is intriguing to examine whether shortened telomeres in somatic nuclei can be restored in nuclear cloning as one aspect of re-juvenilization at the cellular level. A comparison of telomere length among donors, clones and age-matched controls was reported by several groups (). These results indicate that shortened adult cell telomeres can be restored during early development of cloned animals but the degree of telomere elongation is quite variable. Even in a single study, there was significant variation in telomere length among individual clones and among different tissues isolated from a single clone (Betts et al., 2001
; Miyashita et al., 2002
), underscoring the complexity and difficulty of telomere length control in clones. If aborted embryos, which potentially harbor telomeres with extremely abnormal length, are included, the efficiency of telomere restoration may be even less than what is being reported. It is not known whether the telomere elongation in clones is a secondary effect of non-specifically activated telomerase or a regulated telomere restoration reflecting the cell's effort to compensate for defective telomeres.
Telomere length in cloned mammals
To understand the functional consequences of telomere restoration, two groups examined whether the replicative lifespan of senescent cells could be elongated by nuclear cloning. Lanza and colleagues found that clone-derived bovine fibroblast cells, which contained fully restored telomeres comparable to the age-matched controls, showed longer proliferative lifespan than the senescent donor fibroblast cells (Lanza et al., 2000
). However, when Clark and colleagues tested cloned sheep fibroblast cells that harboured partially restored telomeres, the proliferative lifespan of the cells was not extended (Clark et al., 2003
). Thus, it remains to be examined to what extent the restored telomeres can influence the proliferative lifespan of these cells. It is also still unclear whether the resetting of proliferative lifespan of isolated cells has something to do with the lifespan of cloned animals. When whole embryonic extract was tested, the temporal profile of the telomerase activity during development was similar in cloned bovine embryos and fertilized embryos (Xu and Yang, 2001
). Because a number of factors contribute to define the telomerase activity and telomere length in vivo as described above, the next step will be to examine individual accessory factors of the telomerase complex and subnuclear localization of TERT in cloned animals.