Senescence-associated (beta)-galactosidase is widely used as a biomarker of replicative senescence. However, it remains unknown whether this is a distinct enzyme active at pH 6, and differentially expressed in senescence, or a manifestation of an increase in the classic acid lysosomal (beta)-galactosidase. Here we have investigated the origin of senescence-associated-(beta)-galactosidase activity by modifying the intracellular and lysosomal pH of young and senescent human umbilical vein endothelial cells and examining the effect of these manipulations on the levels of activity, using a flow cytometric assay. Lysosomal alkalinisation with chloroquine or bafilomycin A(1), as well as equilibration of the intracellular milieu to pH 6 with nigericin, caused a profound (92-99%) inhibition of the total intracellular (beta)-galactosidase activity. However, independent of pH alterations, senescent cells showed levels of (beta)-galactosidase activity three- to sixfold higher than young cells. This increase in activity occurred in parallel to an increase in (beta)-galactosidase protein levels. Acridine Orange staining revealed an increase in lysosomal content with replicative age, which correlated with the increase in (beta)-galactosidase. These findings demonstrate that senescence-associated (beta)-galactosidase is a manifestation of residual lysosomal activity at a suboptimal pH, which becomes detectable due to the increased lysosomal content in senescent cells.

Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells.

Normal human diploid fibroblasts have a finite replicative lifespan in vitro, which has been postulated to be a cellular manifestation of aging in vivo. Several studies have shown an inverse relationship between donor age and fibroblast culture replicative lifespan; however, in all cases, the correlation was weak, and, with few exceptions, the health status of the donors was unknown. We have determined the replicative lifespans of 124 skin fibroblast cell lines established from donors of different ages as part of the Baltimore Longitudinal Study of Aging. All of the donors were medically examined and were declared “healthy,” according to Baltimore Longitudinal Study of Aging protocols, at the time the biopsies were taken. Both long- and short-lived cell lines were observed in all age groups, but no significant correlation between the proliferative potential of the cell lines and donor age was found. A comparison of multiple cell lines established from the same donors at different ages also failed to reveal any significant trends between proliferative potential and donor age. The rate of [3H]thymidine incorporation and the initial rates of growth during the first few subcultivations were examined in a subset of cell lines and were found to be significantly greater in fetal lines than in postnatal lines. Cell lines established from adults did not vary significantly either in initial growth rate or in [3H]thymidine incorporation. These results clearly indicate that, if health status and biopsy conditions are controlled, the replicative lifespan of fibroblasts in culture does not correlate with donor age.

/pdf/relationship-between-donor-age-and-the-replicative-lifespan-35r9jbfi1q.pdf

Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation.

Werner syndrome is a rare, autosomal recessive condition with multiple progeroid features, but it is a phenocopy of aging rather than accelerated or premature senescence Somatic chromosome aberrations occur in multiple tissues in vivo and in vitro and there is an increased incidence of neoplasia Thus, Werner syndrome can be classified in the group of chromosome instability syndromes Recent findings provide additional support for the concept that there is an aberration of connective tissue metabolism in Werner syndrome, but it is unclear whether this is a primary or secondary manifestation of the underlying genetic defect Abnormal growth characteristics are observed in cultured skin fibroblast-like cells and this provides another avenue for current research Identification of the basic genetic defect in Werner syndrome might clarify our understanding of the normal aging process in general, or might elucidate specific aspects such as the development of neoplasia, atherosclerosis, diabetes, or osteoporosis

Werner's syndrome: a review of recent research with an analysis of connective tissue metabolism, growth control of cultured cells, and chromosomal aberrations.

Impaired DNA damage repair, especially deficient transcription-coupled nucleotide excision repair, leads to segmental progeroid syndromes in human patients as well as in rodent models. Furthermore, DNA double-strand break signalling has been pinpointed as a key inducer of cellular senescence. Several recent findings suggest that another DNA repair pathway, interstrand cross-link (ICL) repair, might also contribute to cell and organism aging. Therefore, we summarize and discuss here that (i) systemic administration of anti-cancer chemotherapeutics, in many cases DNA cross-linking drugs, induces premature progeroid frailty in long-term survivors; (ii) that ICL-inducing 8-methoxy-psoralen/UVA phototherapy leads to signs of premature skin aging as prominent long-term side effect and (iii) that mutated factors involved in ICL repair like ERCC1/XPF, the Fanconi anaemia proteins, WRN and SNEV lead to reduced replicative life span in vitro and segmental progeroid syndromes in vivo. However, since ICL-inducing drugs cause damage different from ICL and since all currently known ICL repair factors work in more than one pathway, further work will be needed to dissect the actual contribution of ICL damage to aging.

/pdf/contributions-of-dna-interstrand-cross-links-to-aging-of-ln9orwalra.pdf

Contributions of DNA interstrand cross-links to aging of cells and organisms

Expression of senescence-associated β-galactosidase (SA-β-Gal) by human skin fibroblasts, effect of advanced glycation end-products and fucose or rhamnose-rich polysaccharides

We have carried out studies on cultured human fibroblasts in an attempt to trace the origins of age-dependent disorders to the cellular and molecular levels. Three interrelated areas are discussed. First, skin donors with diabetes mellitus (a disease complex that features inappropriate hyperglycemia) produce cultured fibroblasts with a moderate reduction in growth capacity, while two inherited disorders of inappropriate hyperglycemia and premature aging, progeria and Werner syndrome, yield fibroblast cultures with more severely impaired growth capacity. Second, there is a decreased response of progeria level and donor age; evidence is presented that this defective hormone responsiveness in aging cells may reside at the hormone receptor on the surface membrane, the cyclic AMP system, the intracellular enzymatic machinery, or all of these sites. Third, tissue factor, a procoagulant that activates the extrinsic clotting mechanism, is more abundant in cells from the premature aging syndromes of progeria and Werner syndrome. Fibroblast aging in vitro may help to explain various concomitants of normal aging and diabetes mellitus including cell dropout, impairment of hormone responsiveness, and increased atherothrombosis.

In vitro studies of age-associated diseases.

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In vitro studies of age-associated diseases.