Samuel Goldstein

Diverse gene sequences are overexpressed in werner syndrome fibroblasts undergoing premature replicative senescence.

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 [32P]cDNA derived from RNA of WS cells compared with [32P]cDNA 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 alpha 1(I) procollagen, alpha 2(I) procollagen, fibronectin, ferritin heavy chain, insulinlike growth factor-binding protein-3 (IGFBP-3), osteonectin, human tissue plasminogen activator inhibitor type I, thrombospondin, and alpha B-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 interpersed nuclear element I (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 [alpha 1(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 early-passage 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.

Regulation of a Novel Gene Encoding a Lysyl Oxidase-related Protein in Cellular Adhesion and Senescence

We report here a novel cDNA clone with a predicted protein sequence similar to lysyl oxidase. This full-length cDNA clone of 3432 base pairs (WS9-14) was isolated from human fibroblasts on the basis of its overexpression in senescent cells. It encodes an 87-kDa polypeptide, whose protein is a member of the scavenger receptor cysteine-rich family, because it contains four scavenger receptor cysteine-rich domains that are found in several secreted or cell surface proteins. The WS9-14 protein has a 48% identity with both lysyl oxidase and lysyl oxidase-like protein at a region corresponding to exons 2–6, implying the existence of a lysyl oxidase gene family. The pattern of WS9-14 gene expression by fibroblasts parallels pro-collagen I-α1 expression. Its mRNA level is induced by transforming growth factor β-1 and indomethacin and inhibited by phorbol ester and retinoic acid. WS9-14 is abundantly expressed in all tumor cell lines examined that attach to culture dishes but not in cell lines that grow in suspension and is also up-regulated in senescent fibroblasts. These results suggest that WS9-14 gene encodes an extracellular protein that may be specifically involved in cell adhesion and senescence.

An overexpressed gene transcript in senescent and quiescent human fibroblasts encoding a novel protein in the epidermal growth factor-like repeat family stimulates DNA synthesis.

We carried out subtractive enrichment of a cDNA library derived from mRNA of senescent human diploid fibroblasts (HDF) established from a subject with Werner syndrome of premature aging. By differential screening, we isolated an overexpressed cDNA sequence (S1-5) that codes for a novel protein containing epidermal growth factor (EGF)-like domains which match the EGF-like consensus sequences within several known extracellular proteins that play a role in cell growth, development, and cell signalling. S1-5 mRNA is overexpressed in Werner syndrome and senescent normal HDF, is induced by growth arrest of young normal cells, but is significantly decreased by high serum, conditions which promote cellular proliferation. Paradoxically, microinjection into young HDF of two different lengths of S1-5 mRNA, containing different putative AUG translational start sites, consistently stimulated rather than inhibited DNA synthesis by an apparent autocrine/paracrine mechanism. Thus, the S1-5 gene product may represent a negative and/or positive factor whose ultimate activity is modulated by the cell environment as occurs with other members of EGF-like family.

The Role of DNA Repair in Aging of Cultured Fibroblasts from Xeroderma Pigmentosum and Normals

Summary Deficient DNA repair in fibroblasts from xeroderma pigmentosum is expressed as markedly reduced survival after low doses of uv. However, the life-span in vitro of XP cells is not reduced below normal age-matched controls. Senescence of normal cells in vitro, measured by decreased plating efficiency, appears before any measurable decline in either the capacity for DNA repair or survival following uv. In no cases was spontaneous transformation to permanent cell lines observed. It is concluded that failure in DNA repair is probably not causal to aging in vitro or in vivo.

Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts

A specific family of tandemly repeated DNA sequences was found to diminish in the human genome after serial passage of three strains of diploid fibroblasts. Eco RI restriction fragments of 340 and 680 bp were significantly reduced in quantity at late passage as determined by autoradiography of 14C-DNA and also by ethidium bromide fluorescence. The reduction in these closely related DNA sequences was confirmed by saturation hybridization to excess 14H-RNA transcribed from a homogeneous restriction fragment recleaved from the 340 bp DNA. The maximal fraction of DNA hybridizing to the 3H-RNA probe declined by 33-50% over 21-41 population doublings. Divergence and/or methylation of such sequences could not account for these results since the thermal stability of cRNA:DNA duplexes actually increased by 0.3 degrees C at late passage. Total highly repetitive sequences assayed by reassociation kinetics were also substantially reduced at late passage, implying that depletion may be common to many repeat families in DNA. The denaturation temperature for such rapidly reassociated duplexes again increased slightly at late passage, possibly reflecting the minor decreases in DNA methylation which were detected in two of the cell strains. Karyotype analyses demonstrated that over 95% euploidy was maintained, with no specific chromosome loss and no visible deletions at late passage. The depletion of reiterated sequences during repeated cell division is thus attributed to numerous small DNA deletions, which may arise from unequal recombination coupled with selection or from a nonreciprocal mechanism such as excision.

Aging in vitro: Growth of cultured cells from the Galapagos tortoise

Abstract Skin biopsies from two growing and two fully-grown Galapagos tortoises, a species that lives twice as long as man, were explanted in vitro. Cellular outgrowth was more vigorous from explants of young tortoises but epithelial cells stopped dividing early in the history of all cultures, even at the optimum temperature of 30 °C. Serially subcultured fibroblasts from young tortoises divided more rapidly and achieved longer lifespans than fibroblasts from old tortoises in terms of mean population doublings and to a lesser extent, calendar time. For all cultures the lifespans exceeded those reported for human diploid fibroblasts. The results indicate that a proportionality exists between the potential (remaining) lifespan in vivo and the mitotic capacity of cultured diploid cells in vitro.

A novel gene encoding a smooth muscle protein is overexpressed in senescent human fibroblasts

In order to identify genes that may be causally involved in replicative senescence, we have isolated several gene sequences that are overexpressed in senescent human fibroblasts by differential screening of a cDNA library derived from mRNA of a subject with Werner syndrome of premature aging (Murano, S., et al., Molec. Cell. Biol., 3905-3914, 1991). Herein, we describe the sequence and expression of one of these genes, WS3-10, which encodes a novel human cytoplasmic protein of 22.5 kilodaltons. The steady-state mRNA levels of WS3-10 mRNA were higher in WS and late-passage normal cells compared to early-passage normal cells following serum depletion and subsequent repletion. Computer analysis showed similarities between WS3-10 and certain proteins in other species, indicating that WS3-10 represents the human homolog of a smooth muscle protein involved in calcium interactions that may contribute to replicative senescence.

Senescence of cultured human fibroblasts: Mitotic versus metabolic time

Abstract To explore the relative importance of mitotic versus non-mitotic (metabolic) factors in determining the finite lifespan of human fibroblasts, mass cultures and 5 clones from 2 normal adults were followed as cohort pairs monitoring calendar time, mean population doublings (MPD), plating efficiency and expression of HL-A antigens. One member of each pair was split serially while the other was held in relative stationary phase with weekly refeeding using standard medium and culture conditions throughout. First cohorts became senescent after 52–62 MPD and 105–170 days while plating efficiencies declined and some HL-A antigens lost reactivity with specific antisera. At this time second cohorts were released from stationary phase and split serially. After initial loss of viability they generally showed higher plating efficiencies and normal reactivity of HL-A antigens till cell death within a calendar time of 195–240 days. The total MPD were reduced only marginally if allowance was made for low grade mitosis occurring during prolonged stationary phase. The results indicate that continuously replicating cells lose viability significantly before mitotically inhibited but actively metabolizing cohorts and suggest that factors which increase cellular turnover accelerate senescence and its pathological sequelae.

Heat-Labile Enzymes in Skin Fibroblasts from Subjects with Progeria

To characterize further the genetic basis of progeria, thermolability studies were performed on three genetically distinct enzymes in crude extracts of cultured skin fibroblasts derived from two subjects with that syndrome. At early passage the progeric fibroblasts, as compared to controls, contained a significantly higher percentage of heat-labile glucose-6-phosphate dehydrogenase (12.83 plus or minus 1.72 vs 1.11 plus or minus 0.44 [mean plus or minus S.E.M.], p smaller than 0.001), 6-phosphogluconate dehydrogenase (9.71 plus or minus 0.68 vs. 0.67 plus or minus 0.22, p smaller than 0.001), and hypoxanthine-guanine phosphoribosyltransferase (31.41 plus or minus 1.89 vs 7.67 plus or minus 1.71, p smaller than 0.001), and the differences were maintained throughout the in vitro life-span. These data, in conjunction with previous reports of defective HL-A antigens, indicate a widespread defect in genetic expression. The most likely cause appears to be an aberration in protein synthesis or degradation, or both, although multiple somatic mutations cannot be ruled out. Increased thermolability of enzymes in cultured cells may provide a screening test for persons predisposed to progeria and other disorders of premature aging.

Cancer in the Elderly: Basic Science and Clinical Aspects

The incidence of cancer increases progressively with age. Rearrangements of genomes have been found to accompany cellular aging. These factors, in concert with age-dependent alterations in immune function and host defense, may help to explain the increased risk of malignant disease in aged persons. The clinical presentation and natural history of neoplasia are also affected by aging. This conference reviews recent developments in these areas, examines the effects of drug use in the elderly and implications for management, and discusses current information on how age may influence the response of cancer to therapy.