The potential of cloning depends in part on whether the procedure can reverse cellular aging and restore somatic cells to a phenotypically youthful state. Here, we report the birth of six healthy cloned calves derived from populations of senescent donor somatic cells. Nuclear transfer extended the replicative life-span of senescent cells (zero to four population doublings remaining) to greater than 90 population doublings. Early population doubling level complementary DNA-1 (EPC-1, an age-dependent gene) expression in cells from the cloned animals was 3.5- to 5-fold higher than that in cells from age-matched (5 to 10 months old) controls. Southern blot and flow cytometric analyses indicated that the telomeres were also extended beyond those of newborn (<2 weeks old) and age-matched control animals. The ability to regenerate animals and cells may have important implications for medicine and the study of mammalian aging.

Animals↗

PubMed2000

Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer.

Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan < 100 animals) were electrofused with enucleated oocytes from domestic cows. Twelve percent of the reconstructed oocytes developed to the blastocyst stage, and 18% of these embryos developed to the fetal stage when transferred to surrogate mothers. Three of the fetuses were electively removed at days 46 to 54 of gestation, and two continued gestation longer than 180 (ongoing) and 200 days, respectively. Microsatellite marker and cytogenetic analyses confirmed that the nuclear genome of the cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations.

Alleles↗

PubMed1999

Human therapeutic cloning.

Somatic cell nuclear 'reprogramming' in livestock species is now routine in many laboratories. Here, Robert Lanza, Jose Cibelli and Michael West discuss how these techniques may soon be used to clone genetically matched cells and tissues for transplantation into patients suffering from a wide range of disorders that result from tissue loss or dysfunction.

Animals↗

PubMed1999

Prospects for the use of nuclear transfer in human transplantation.

The successful application of nuclear transfer techniques to a range of mammalian species has brought the possibility of human therapeutic cloning significantly closer. The objective of therapeutic cloning is to produce pluripotent stem cells that carry the nuclear genome of the patient and then induce them to differentiate into replacement cells, such as cardiomyocytes to replace damaged heart tissue or insulin-producing beta cells for patients with diabetes. Although cloning would eliminate the critical problem of immune incompatibility, there is also the task of reconstituting the cells into more complex tissues and organs in vitro. In the review, we discuss recent progress that has been made in this field as well as the inherent dangers and scientific challenges that remain before these techniques can be used to harness genetically matched cells and tissues for human transplantation.

Cloning, Organism↗

PubMed1999

Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells.

PURPOSE: To investigate the relation of senescence-related beta-galactosidase activity and telomere shortening to replicative senescence in cultured human retinal pigment epithelial (RPE) cells. METHODS: A human RPE cell line was serially passaged until 80% of cells were nondividing in a 72-hour 5-bromo-2'-deoxyuridine (BrdU) labeling study. Early- and late-passage cells were double-stained for BrdU and senescence-related beta-galactosidase activity (pH 6). The average chromosomal telomere length at several population doublings was estimated by Southern blot analysis after double digestion of DNA with RsaI and HinfI and using a telomere-specific probe. RESULTS: BrdU-beta-galactosidase double-staining revealed an inverse correlation between the number of BrdU-labeled nuclei and beta-galactosidase-labeled cells as a function of population doubling level (PDL). At PDL 58, only 20% of all cells labeled for BrdU, whereas 57% stained for beta-galactosidase. The mean terminal restriction fragment length (TRF) was reduced from 10 kb in early (PDL 12) cultures to 4 kb in late (PDL 57) cultures. CONCLUSIONS: Senescence-related beta-galactosidase activity and mean TRF length may prove useful in studying the senescence of RPE cells in vitro. These techniques may be valuable in determining senescence of the retinal pigment epithelium in vivo, where senescent RPE cells could be involved in the development of age-related maculopathy and age-related macular degeneration.

Blotting, Southern↗

PubMed1997

ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase.

Telomere loss has been proposed as a mechanism for counting cell divisions during aging in normal somatic cells. How such a mitotic clock initiates the intracellular signalling events that culminate in G1 cell cycle arrest and senescence to restrict the lifespan of normal human cells is not known. We investigated the possibility that critically short telomere length activates a DNA damage response pathway involving p53 and p21(WAF1) in aging cells. We show that the DNA binding and transcriptional activity of p53 protein increases with cell age in the absence of any marked increase in the level of p53 protein, and that p21(WAF1) promoter activity in senescent cells is dependent on both p53 and the transcriptional co-activator p300. Moreover, we detected increased specific activity of p53 protein in AT fibroblasts, which exhibit accelerated telomere loss and undergo premature senescence, compared with normal fibroblasts. We investigated the possibility that poly(ADP-ribose) polymerase is involved in the post-translational activation of p53 protein in aging cells. We show that p53 protein can associate with PARP and inhibition of PARP activity leads to abrogation of p21 and mdm2 expression in response to DNA damage. Moreover, inhibition of PARP activity leads to extension of cellular lifespan. In contrast, hyperoxia, an activator of PARP, is associated with accelerated telomere loss, activation of p53 and premature senescence. We propose that p53 is post-translationally activated not only in response to DNA damage but also in response to the critical shortening of telomeres that occurs during cellular aging.

Alleles↗

PubMed1996

Shortened telomeres in the expanded CD28-CD8+ cell subset in HIV disease implicate replicative senescence in HIV pathogenesis.

OBJECTIVE: To test the hypothesis that the expanded population of non-proliferative CD28-CD8+ T cells in HIV disease have shortened telomeres, thereby providing evidence that increased rounds of CD8+ cell division occur during HIV disease, possibly leading to replicative senescence and exhaustion of CD8+ T-cell responses. DESIGN: CD8+ cells play a central role in control of HIV infection. In late HIV disease, an expanded population of CD28-CD8+ cells with reduced proliferative potential has been documented. A similar population of CD28-CD8+ cells has been identified in ageing humans, where telomere length measurements have suggested that these cells have reached the irreversible state of replicative senescence. METHODS: CD8+ cells from HIV-infected and control subjects were sorted by flow cytometry into CD28+ and CD28- fractions. Telomere lengths were determined as mean terminal restriction fragment (TRF) lengths by Southern hybridization. RESULTS: The TRF lengths of sorted CD28-CD8+ cells in HIV-infected subjects ranged between 5 and 7 kilobases (kb) and were significantly shorter than TRF lengths of CD28-CD8+ cells in uninfected subjects (P = 0.003). The TRF length in CD28-CD8+ cells from HIV-infected subjects was the same as that observed for centenarian peripheral blood mononuclear cells and is compatible with a state of replicative senescence. CONCLUSIONS: The shortened telomeres in the CD28-CD8+ cells in HIV-infected subjects and the poor proliferative potential of these cells identifies CD8+ cell replicative senescence as a newly described feature of HIV disease. Our results provide a mechanism for the loss of CD8+ cell control of viral replication that accompanies advanced HIV disease. Replicative senescence may contribute to exhaustion of the T-cell response as a result of chronic HIV disease. Whether this phenomenon occurs in other chronic viral infections is unknown.

CD28 Antigens↗

PubMed1995

Replicative senescence and cell death.

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Animals↗

PubMed1995

Brugia pahangi: differential granulomatous reactivity of infected jirds (Meriones unguiculatus) to fractions of adult worm extract.

Soluble extracts of adult Brugia pahangi (SSE) were fractionated by lectin affinity chromatography, followed by reversed phase HPLC. The immunologic and in vivo inflammatory reactivity of the resulting fractions were compared in jirds with acute and chronic infections of B. pahangi. When separated by SDS-PAGE, all fractions possessed bands which were recognized in Western blots by antibodies from jirds with both acute and chronic infections. Fractions were coupled to sized Sepharose beads that were subsequently embolized into the lungs of infected and uninfected control jirds. Granulomas were induced by SSE, the lectin column eluate, and HPLC fractions E, F, and G in acutely infected jirds. These reactions were significantly reduced in chronically infected jirds. HPLC fractions B, C, and D did not elicit an in vivo inflammatory response. A perivascular infiltrate of eosinophils and mononuclear cells was also observed in lungs of acutely infected jirds which received granuloma-inducing coated beads but not in lungs of similar jirds which received beads that did not induce this inflammatory response. Proliferative responses of splenocytes stimulated with SSE or the lectin eluate and lymph node cells and splenocytes stimulated with HPLC fractions B, C, or D corresponded to the in vivo granulomatous response to homologously coated beads. Correlations between in vivo inflammatory responses and in vitro proliferative responses were not seen using other fractions in these assays. These data indicate that varying degrees of granulomatous inflammation are induced by different filarial proteins mixtures and that the in vivo granuloma induction by antigen-coated beads will be useful in the identification of specific proteins involved in the induction, maintenance, and regulation of filariae-elicited inflammatory reactions. Although the size of these granulomas corresponds to severity of granulomatous inflammatory responses visualized within the jird lymphatics during the course of infection, the reaction does not correlate in all instances to lymphoproliferative responses of cells from peripheral lymph nodes or the spleen. Distinct differences between antibody and granulomatous reactivity to some fractions were noted.

Animals↗

PubMed1995

Aspects of the workplace and return to work for persons with brain injury in supported employment.

This prospective study examined the effect of work environments on return to work for persons with brain injuries. Participants (n = 37) were individuals placed into supported employment by six placement agencies. All were assessed using the Vocational Integration Index (VII), an observational instrument for rating the opportunities for integration (Job Scale) and the extent to which an employee benefits from those opportunities (Consumer Scale). Individuals who retained their jobs for 6 months (n = 19) had been rated higher on all subscales and total scores for the VII, with seven of eight subscales statistically significant. Findings are discussed in regard to improving employment outcomes for persons with severe brain injuries.

Adult↗

PubMed1995

Common sense is alive and well: comments on Erb (1995)

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Community Mental Health Services↗

PubMed1994

Specific association of human telomerase activity with immortal cells and cancer.

Synthesis of DNA at chromosome ends by telomerase may be necessary for indefinite proliferation of human cells. A highly sensitive assay for measuring telomerase activity was developed. In cultured cells representing 18 different human tissues, 98 of 100 immortal and none of 22 mortal populations were positive for telomerase. Similarly, 90 of 101 biopsies representing 12 human tumor types and none of 50 normal somatic tissues were positive. Normal ovaries and testes were positive, but benign tumors such as fibroids were negative. Thus, telomerase appears to be stringently repressed in normal human somatic tissues but reactivated in cancer, where immortal cells are likely required to maintain tumor growth.

Base Sequence↗

PubMed1994

Telomerase, cell immortality, and cancer.

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Antineoplastic Agents↗

PubMed1994

The cellular and molecular biology of skin aging.

BACKGROUND: The dramatic alterations in the appearance of the integument with increasing age are due in part to a progressive destruction of the delicate architecture of the connective tissue components of the dermis. Both collagenous and elastic components display a degeneration consistent with the overexpression of proteolytic activity. Recent advances in the field of molecular gerontology, using in vitro models of cellular aging, are yielding clues as to the fundamental causes of dermal aging. OBSERVATIONS: Dermal fibroblasts possess a finite replicative capacity of 50 to 100 doublings, then cease replicating in response to growth factors. Cells cultivated to the end of their replicative lifespan in vitro display alterations consistent with their playing a role in aging in vivo. In particular, senescent dermal fibroblasts overexpress metalloproteinase activities that may explain the age-related atrophy of extracellular matrix architecture. CONCLUSIONS: The recent discovery of a structural change in the telomeric region of the genome with cellular aging and new insights into DNA damage checkpoint mechanisms offer new opportunities to uncover both the molecular mechanisms regulating cellular aging and possibly to devise new strategies to manipulate these molecular events for therapeutic effect.

Cellular Senescence↗