Novel monoclonal antibodies identify antigenic determinants unique to cellular senescence

Abstract

Normal human diploid fibroblasts exhibit a limited lifespan in vitro and are used as a model to study in vivo aging. Monoclonal antibodies were generated against partially purified surface membranes from human diploid fibroblasts at the end of their lifespan (senescent). Three hybridomas were isolated that secreted antibodies reacting to cellular determinants expressed specifically on senescent human fibroblasts of different origin, including neonatal foreskin, embryonic lung, and adult skin punch biopsy, but not expressed on matched young cells. The antibodies did not bind to immortal human cells and normal young cells made reversibly nondividing, indicating the antigens are not expressed in cells that are not senescent. The antibodies identified senescent cells in a mixed cell population and expression of the senescent cell antigens correlated strongly with the cells inability to synthesize DNA at the onset of senescence. The antigens appeared to be cell surface or extracellular matrix associated, and the epitopes were destroyed by mild trypsin treatment. Western analysis indicated all three antibodies reacted with fibronectin. Though the antigenic determinants on the fibronectin molecule were not accessible in the intact young cell, the epitopes were present in fibronectin extracted from both senescent and young cells, as well as purified human plasma fibronectin. These antibodies and the senescent specific expression of the antigens provide powerful tools to investigate the mechanisms leading to in vitro senescence. This may enable us to investigate directly the relationship between cellular aging and aging of the individual.

A unique, short sequence determines p53 gene basal and UV-inducible expression in normal human cells

“…Six normal human diploid ®broblast (NHDF) cell lines: HCA2 and Jas3, described in our previous studies (Noda et al, 1994;Porter et al, 1990), and WI26, TIG3, TIG7 and MRC5, provided by the Japanese Cancer Research Resources Bank (JCRB), were used for the p53-promoter analysis. T98G, a human glioblastoma cell line with homozygous mutant p53 gene status (at codon 237, Met to Ile, Ullrich et al, 1992), and HeLa S3, a human cervical carcinoma cell line, were also obtained from JCRB.…”

Modifications of proteoglycans secreted into the growth medium by young and senescent human skin fibroblasts

“…Quantitative and qualitative changes in the synthesis of certain proteins [5][6][7][8] suggest that both efficiency and specificity of the biochemical machinery for protein synthesis may be different in senescent cells. These changes also affect the main components of extracellular matrix (ECM), such as fibronectin [9,10] and collagen [5,11]. We have found that the biosynthesis of proteoglycans (PG) is also modified with senescence [12], since late passage HFs incorporate greater amounts of radioactivity in the large PGs of the cell layer and show a lower ratio between PGs containing dermatan sulphate (DS) and those containing heparan sulphate (HS) [12].…”

Modifications of proteoglycans extracted from monolayer cultures of young and senescent human skin fibroblasts

“…In addition to the loss of proliferative capacity, senescent cells display a number of phenotypic changes, including an increase of cell size [5]and variations of extracellular matrix (ECM) components [6–9]. With reference to proteoglycans (PGs), senescent human skin fibroblasts (HFs) release into the growth medium an increased proportion of decorin and secrete PGs containing galactosaminoglycuronan (GalN‐GAG) moieties with a higher sulphation degree of galactosamine residues and a lower percentage content of totally unsulphated polysaccharide chains [10].…”