When human fibroblasts from different donors are grown in vitro, only a small fraction of the variation in their finite replicative capacity is explae by the chronological age of the donor. Because we had previously shown that telomeres, the terminal guanine-rich sequences of chromosomes, shorten throughout the life-span of cultured cells, we wished to determine whether variation in initial telomere length would account for the unexplained variation in replicative capacity. Analysis of cells from 31 donors (aged 0-93 yr) indicated relatively weak correlations between proliferative ability and donor age (m = -0.2 doubling per yr; r = -0.42; P = 0.02) and between telomeric DNA and donor age (m = -15 base pairs per yr; r = -0.43; P = 0.02). However, there was a stiking correlation, valid over the entire age range of the donors, between replicative capacity and initial telomere length (m = 10 doublngs per kilobase pair; r = 0.76; P = 0.004), indicating that cell strains with shorter telomeres underwent slglcantiy fewer doublings than those with longer telomeres. These observations suggest that telomere length is a biomarker ofsomatic cell aging in humans and are consistent with a causal role for telomere loss in this process. We also found that fibroblasts from Hutchinson-Gilford progeria donors had short telomeres, consistent with their reduced division potential in vitro. In contrast, telomeres from sperm DNA did not decrease with age ofthe donor, suggesting that a mechanism for maintaining telomere length, such as telomerase expression, may be active in germ-line tissue.The cellular senescence model of aging was founded by landmark experiments of Hayflick and Moorhead (1), who firmly established that normal human fibroblasts have a finite life-span in vitro. Although much evidence supports this model (2-8), the mechanism accounting for the finite division capacity of normal somatic cells remains a mystery. Olovnikov (9, 10) suggested that the cause of cellular senescence is the gradual loss of telomeres due to the end-replication problem-i.e., the inability of DNA polymerase to completely replicate the 3' end of linear duplex DNA (11) (for review, see refs. 12 and 13).Telomeres play a critical role in chromosome structure and function. They prevent aberrant recombination (14-16) and apparently function in the attachment ofchromosome ends to the nuclear envelope (17 (31)(32)(33). These observations have led to the telomere hypothesis of cellular aging (13), in which loss of telomeres due to incomplete DNA replication and absence of telomerase provides a mitotic clock that ultimately signals cell cycle exit, limiting the replicative capacity of somatic cells. To further explore this hypothesis, we have examined the relationship between telomere length, in vivo age, and replicative capacity of fibroblasts from normal donors and subjects with the Hutchinson-Gilford syndrome of premature aging (34,35). We have also determined the relationship between telomere length in sperm DNA and donor age.MATERIALS AND MET...

Human diploid fibroblasts undergo replicative senescence predominantly because of arrest at the G1/S boundary of the cell cycle. Senescent arrest resembles a process of terminal differentiation that appears to involve repression of proliferation-promoting genes with reciprocal new expression of antiproliferative genes, although post-transcriptional factors may also be involved. Identification of participating genes and clarification of their mechanisms of action will help to elucidate the universal cellular decline of biological aging and an important obverse manifestation, the rare escape of cells from senescence leading to immortalization and oncogenesis.

Genes that play a role in the senescent arrest of cellular replication are likely to be overexpressed in human diploid fibroblasts (HDF) derived from subjects with Werner syndrome (WS) because these cells have a severely curtailed replicative life span. To identify some of these genes, a cDNA library was constructed from WS HDF after they had been serum depleted and repleted (5 days in medium containing 1% serum followed by 24 h in medium containing 20% serum). Differential screening of 7,500 colonies revealed 102 clones that hybridized preferentially with [32PJcDNA derived from RNA of WS cells compared with [32PJcDNA derived from normal HDF. Cross-hybridization and partial DNA sequence determination identified 18 independent gene sequences, 9 of them known and 9 unknown. The known genes included oal(I) procollagen, a2(I) procollagen, fibronectin, ferritin heavy chain, insulinlike growth factor-binding protein-3 (IGFBP-3), osteonectin, human tissue plasminogen activator inhibitor type 1, thrombospondin, and aB-crystallin. The nine unknown clones included two novel gene sequences and seven additional sequences that contained both novel segments and the Alu class of repetitive short interspersed nuclear elements; five of these seven Alu+ clones also contained the long interspersed nuclear element 1 (KpnI) family of repetitive elements. Northern (RNA) analysis, using the 18 sequences as probes, showed higher levels of these mRNAs in WS HDF than in normal HDF. Five selected mRNAs studied in greater detail [al(I) procollagen, fibronectin, insulinlike growth factor-binding protein-3, WS3-10, and WS9-14] showed higher mRNA levels in both WS and late-passage normal HDF than in earlypassage normal HDF at various intervals following serum depletion/repletion and after subculture and growth from sparse to high-density confluent arrest. These results indicate that senescence of both WS and normal HDF is accompanied by overexpression of similar sets of diverse genes which may play a role in the senescent arrest of cellular replication and in the genesis of WS, normal biological aging, and attendant diseases.Biological aging, an inevitable process in multicellular organisms, features two sets of interdependent events: functional decline and an exponential rise in the incidence of degenerative and neoplastic diseases (27). The mechanism of aging remains unknown, but it seems clear that the progressive loss of cellular replicative capacity in many tissues is inextricably involved (26, 27, 51, 55