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I.R.F. / Aging news / Cloning / 03052201

Telomeres Reconstitution in Cloning
Posted on: May 22, 2003

The production of viable, fertile cloned animals by nuclear transplantation of somatic cells from cultured cell lines and adult tissues has challenged the understanding of terminal cell differentiation, cellular aging, and the proliferative capacity of cells. Successful cloning requires the reprogramming of the donor nuclei from pluripotent or differentiated cells to an undifferentiated state to permit the temporal and spatial reexpression of genes involved in embryo and fetal development. Somatic nuclei progressively acquire differentiated functions through the gradual implementation of epigenetic chromatin modifications during embryogenesis and postembryonic development. Amazingly, the cytoplasm of the mammalian oocyte under optimal conditions can reverse the changes to the chromatin structure and function of the differentiated transplanted nucleus to a state of totipotency. The inefficiency in the nuclear reprogramming has been suggested as one of the major causes of the high rate of embryonic, fetal, and neonatal failures observed after nuclear transplantation. One characteristic structural DNA change of most dividing in vivo and in vitro somatic cells is the shortening of the telomeres, the long tandem arrays of hexameric DNA sequences (TTAGGG)n at the ends of mammalian chromosomes, during DNA replication. Telomeres are critical structures that function in the stability, replication, and segregation of the chromosome during mitosis, and the gradual loss of telomeric sequence has been proposed as a "mitotic clock" leading to cell cycle arrest or cellular senescence. Conventional DNA polymerases cannot replicate the extreme 59 ends of chromosomes because removal of the most terminal RNA primer in the lagging strand leaves a small region of uncopied DNA. This telomeric DNA loss has been calculated to be between 50 and 200 bp per cell division. Once telomeres shorten below a critical length, they lose the capacity to cap chromosomes effectively and are thought to activate a DNA damage response pathway that causes cell cycle arrest. Cells can overcome this "end-replication problem" by the Activation and expression of the ribonucleoprotein telomerase, which synthesizes TTAGGG repeats de novo onto the ends of chromosomes. This multisubunit, reverse transcriptase, uses its RNA component as a template for the synthesis of telomeric DNA. Normal human somatic cells do not express telomerase activity and therefore have a limited replicative life span in vitro. Telomerase activity and maintenance of telomere length has been observed in the germ line and in most immortal cell lines and tumor samples analyzed thus far. The introduction of the telomerase catalytic subunit (hTERT) into normal human diploid cells displayed normal growth controls and normal karyotypes and succeeded in extending the life span of the cells. Late-generation mice lacking the telomerase RNA (mTR-/-) component displayed shortened telomeres and chromosome abnormalities and exhibited defective spermatogenesis, increased apoptosis, and decreased proliferation in the testis, bone marrow, and spleen. Thus, telomerase activity in the germ line is thought to prevent cumulative telomere shortening from generation to generation. The telomere hypothesis of aging can be tested in vivo by the use of nuclear transfer technology because it allows the production of cloned animals from adult and cultured somatic cells, without the involvement of the germ line. Sheep cloned by nuclear transfer of cultured cells from embryonic or fetal tissue showed telomere shortening of approximately 10-15% compared with age-matched controls. Furthermore, Dolly, cloned from a cultured mammary cell from a 6-year-old ewe, and displayed telomere loss of approximately 20%. Therefore, both the age of the donor nucleus and the proliferation in culture contributed to the telomeric loss observed in this small sample size of cloned sheep. In contrast, Advanced Cell Technology (ACT) scientists have demonstrated the reversal of cellular aging with the use of senescent donor somatic cells as the nuclear donor. Fibroblasts from bovine fetuses cloned from these cells displayed an extended replicative life span and rebuilding of telomere length compared with control fetal fibroblasts (FFs) and senescent fibroblasts, respectively. Moreover, nucleated blood cells from 5- to 10-month-old cloned cattle appeared to have longer telomere lengths compared with newborn and age-matched control animals. So during several studies on telomere reconstitution in cloned calves it could be stated that telomere loss occurs in cultured bovine fibroblasts and ES-like cells; however, cloned cattle, reconstructed from cultured adult and fetal somatic cells, displayed telomere lengths similar to those of age matched control animals. The rebuilding of telomeric sequences from shorter telomere lengths observed in the donor cells could be due to the presence of telomerase activity, detected as early as the first week of post cloning embryonic development. Reprogramming of telomerase activity was observed as early as the blastocyst stage in nuclear transfer bovine embryos that were reconstructed with the use of various donor nuclei that displayed low and nondetectable levels of telomerase. This appearance of telomerase activity in cloned embryos was delayed, compared with fertilization-derived bovine embryos, in which relatively high levels of telomerase activity was observed after activation of the embryonic genome at the 8‐16-cell stage. Other studies have examined structural and functional reprogramming events in reconstructed embryos, including nucleolar and mitochondrial morphology, nuclear swelling, and the exchange of somatic histone H1 subtypes. These studies have demonstrated that nuclear reprogramming took place over several cell cycles and may be delayed or incomplete in the transferred nucleus. Recently, a differential display analysis that compared cDNA profiles between nuclear-transferred bovine blastocysts with in vivo- and in vitro-derived blastocysts demonstrated that most but not all of the mRNAs were reprogrammed in cloned embryos. Specific epigenetic DNA modifications were probably required for proper transcriptional activation of the embryonic genome. The lack of complete genetic reprogramming of gene expression and chromatin structure may have lead to the developmental failures and abnormalities observed in cloned embryos, fetuses, and offspring. The telomere loss observed in donor somatic cells has been attributed to the age of the donor animal and culture propagation of the cells in vitro. Scientific reports have demonstrated decreased telomerase expression in dividing stem cells in culture, with an associated shortening of telomere lengths. It has been suggested that the observed telomerase activity in candidate bovine stem cells was not sufficient to prevent telomere shortening. Single-stranded telomeric DNA damage, caused by oxygen free radicals, has been shown to accumulate in cells after prolonged periods of culture and confluency, what has lead to an increased rate of telomeric shortening in fibroblasts. This oxidative stress-induced telomeric damage has also been observed in telomerase-positive cells grown in culture. The reduced telomerase activity levels observed in late-passage bovine ES-like cells may have repaired telomeric damage but did not prevent the telomere shortening produced by the "end replication problem." Early-passage cells cultured under serum starvation conditions may also were susceptible to increased telomeric damage and shortening because of reduced telomerase levels due to cell cycle exit into the quiescent (Go) state. Talking about multiple tissue and cell samples from cloned bovine fetuses and newborn calves derived from cultured fetal and adult cell lines, telomere rebuilding was observed there. The mean TRF lengths of nuclear transfer bovine fetuses and offspring was not significantly different from those of age-matched control samples. However, the animal-to-animal variation and tissue-to-tissue variation between and within newborn cloned calves, was found respectively. Some studies have shown significant telomere length differences among individuals, and the telomere synchrony observed in fetal tissues was lost during postnatal life. The telomere length variations among and within newborn calves could be due to donor cell selection, tissue and animal-specific differences in telomere rebuilding, and/or the different proliferative rates of different tissues. Comparing cloned calves and those of ACT results; they are in contrast to observations on cloned sheep, which were shown to have shortened telomeres. Despite having shorter mean TRF (telomere repeat fragments) lengths, nuclear-transfer sheep were healthy, fertile, and typical for sheep of their breeds. The birth of cloned offspring, their development to adulthood strongly suggested that proliferative capacity of late-passage tissue culture cells, and cells from very old adult animals could be restored to a considerable degree by nuclear transplantation. However, it has not been determined whether telomere length accurately reflects the physiological age of an animal. Mice deficient in telomerase activity only showed a disrupted phenotype after 4-5 generations, and the sequential cloning of mice by transfer of adult cumulus cell nuclei showed no adverse effects. It should be noted that mice telomeres are substantially longer than those of other species, and telomerase activity is present in most of their adult somatic cells. Talking about telomere length the possibility that there are also other regulatory mechanisms other than just the presence of telomerase activity shouldn't be rejected. Each vertebrate species has a set characteristic maximum telomere length, and many immortalized and cancer cell lines where telomerase has been reactivated showed extremely high levels of telomerase activity that only maintains short telomeres. Telomere specific binding proteins such as telomeric repeat binding factor- 1, telomeric repeat binding factor-2, tankyrase, and telomeric repeat binding factor-1 interacting nuclear protein-2 have recently been implicated as mediators of telomere length. These proteins may directly inhibit/facilitate the binding of telomerase to telomeric DNA or provide structural changes within the telomere that prevent/promote telomerase binding. Interestingly, telomeric repeat binding factor-2 is up regulated in senescent human fibroblasts. Complex telomere remodeling and telomerase regulation during nuclear reprogramming may have promoted telomere restoration and possibly telomere length extension in cloned offspring. Alternatively, because most of the cloned calves studied ACT experienced pulmonary hypertension, respiratory distress and fever before 4 months of age, an activated immune response might have triggered an up-regulation of telomerase activity and subsequent lengthening of the telomeres in nucleated blood cells. In summary, the reprogramming of telomerase activity and the restoration of telomere length in cloned cattle derived from the nuclear transfer of cultured and aged somatic cells phenomenon exists. The telomere rebuilding observed in cloned cattle may be attributed to the nuclear reprogramming of telomerase activity that was detected at the blastocyst stage of cloned embryo development. Detection of telomerase reexpression could be used as a marker to assess the extent and timing of nuclear reprogramming in reconstructed embryos. Such observations demonstrate that cloned offspring repair genomic modifications acquired during the donor nuclei's in vivo and in vitro period before nuclear transfer, suggesting that along with telomere shortening, nuclear reprogramming outside of the germ line may repair other forms of DNA alterations, such as DNA damage. Telomere research on cloned mammals may determine the mechanism and timing of telomere restoration and any physiological effects of somatic cell nuclear transfer on the aging process.

Source: Dean H. Betts, Vilceu Bordignon, Jonathan R. Hill, Quinton Winger, Mark E. Westhusin, Lawrence C. Smith, and W. Allan King; Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle; PNAS, January 30, 2001, vol. 98, No.3, 1077-1082.
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