Ray Thweatt

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.

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.

Insulin-like growth factor binding protein-3 is overexpressed in senescent and quiescent human fibroblasts.

Cellular insulin-like growth factor binding protein-3 (IGFBP-3) mRNA and IGFBP-3 levels in conditioned medium were consistently higher in cultures of late passage normal (old) fibroblasts and prematurely senescent fibroblasts derived from Werner syndrome (WS) during quiescence induced by serum depletion and during the renewed growth ensuing after serum repletion, compared to cultures of early passage normal (young) fibroblasts. Molar ratios of IGFBP-3/IGF-II were always higher in senescent cultures and maintained a hierarchy of old > WS > young human diploid fibroblasts. Transfection into fibroblasts of the normal full-length IGFBP-3 cDNA in an expression vector resulted in a significant reduction in colony formation compared to cells transfected with an empty expression vector (no cDNA) or with IGFBP-3 cDNA altered by a 273 base pair (bp) deletion. Addition to old and young cultures of recombinant human IGFBP-3 and IGF-I at 1:1 or 5:1 molar ratios inhibited IGF-I-mediated DNA synthesis by approximately 70-80%. These data indicate that IGFBP-3 may play an important role in the quiescent and senescent growth arrest of HDF.

Studies on the molecular-genetic basis of replicative senescence in Werner syndrome and normal fibroblasts

Based on evidence that human diploid fibroblasts (HDF) from the Werner syndrome (WS) of premature aging might overexpress an inhibitor of DNA synthesis (IDS), we prepared a eukaryotic cDNA expression library from WS mRNA and tested it for IDS activity in a transient assay. Two of six WS cDNA pools tested gave IDS activity, then on plus/minus screening revealed several differentially expressed cDNA clones. By slot blot and Northern analysis, one cDNA clone was found to be overexpressed in WS and normal senescent HDF, but not in quiescent normal HDF, indicating that it is senescence-specific. Further studies are needed to clarify: a) whether this cDNA truly acts as an IDS; b) if so, whether it acts alone or in concert with other cDNAs; and c) whether it is involved in the degenerative and malignant sequelae of WS and normal aging.

Characterization Of IGFBP-3, PAI-1 and SPARC mRNA expression in senescent fibroblasts

The RNA species encoded by IGFBP-3 (insulin-like growth factor binding protein-3), PAI-1 (plasminogen activator inhibitor-1) and SPARC (secreted protein-acidic and rich in cysteine; a.k.a. osteonectin) are overexpressed in senescent human diploid fibroblasts (HDF). Their extracellular products have the ability to modulate cell growth in culture and have been shown to have inhibitory effects on DNA synthesis and/or cell growth. This overproduction may contribute to a number of features of aging, including osteoporosis, atherosclerosis and diabetes mellitus type II. Based on analysis of steady-state mRNA levels, which showed similar patterns for all three along with overexpression in senescent cells, we further investigated their transcription rates and stability to determine reasons for their overexpression and to determine if coordinate gene regulation was involved. Characterization of the rates of transcription and the levels of message stability of these genes in early passage (young) versus late passage (old) HDF revealed that IGFBP-3, PAI-1 and SPARC are coordinately overexpressed but not regulated by a unique or simple mechanism encompassing all three transcripts. Only PAI-1 shows an increase in the rate of transcription, while all three show evidence that their overexpression is due to an increase in the stability of RNA. Thus, the overexpression of these genes in senescent fibroblasts involves interactions not only at the transcriptional level but also with protein factors involved in determining the stability and the degradation of RNA.

Expression of senescence-induced protein WS3-10 in vivo and in vitro

In our efforts to characterize cellular senescence we have shown that the mRNA encoding WS3-10 protein is overexpressed in senescent human diploid fibroblasts (HDF) when compared with their younger counterparts, and that forced expression of the WS3-10 cDNA in young HDF results in suppression of calcium-dependent membrane currents, presumably due in part to the presence of a calcium binding domain within the WS3-10 protein. We have now expressed this protein in E. coli and have obtained affinity purified antibodies. Western blot analysis utilizing these antibodies showed that WS3-10 protein is also overexpressed in senescent HDF when compared to young HDF, and in normal fetal lung HDF when compared to SV40-transformed fetal lung HDF. HeLa cells do not express WS3-10 protein. In addition, we looked for WS3-10-related species in a variety of rat tissues. Analysis of WS3-10 immunologically related proteins in rat tissue extracts revealed two WS3-10 homologs, sized 22 kDa and 20 kDa. The latter presumably result from proteolytic removal of the C-terminal end of the 22 kDa polypeptide. The ratio between these polypeptides varies in a tissue-specific manner. Two proteins immunologically related to WS3-10 with sizes of 39 kDa and 91 kDa were present in rat spleen and skeletal muscle, respectively.

Derangements in Calcium‐Dependent Membrane Currents in Senescent Human Fibroblasts Are Associated with Overexpression of a Novel Gene Sequencea

Biological aging in metazoans is accompanied by functional decline and a rising incidence of degenerative and neoplastic diseases.’ Although the mechanism(s) remains unknown, the progressive loss of cellular replicative capacity in several tissues is intimately involved.” Human diploid fibroblasts (HDF) possess a limited replicative lifespan which can be monitored by the cumulative number of mean population doublings (MPD) before ~enescence.~.~ The maximum MPD attained by a variety of HDF cultures show an inverse proportion to donor age, and HDF derived from individuals with inherited disorders that display premature aging, such as Werner syndrome (WS), show premature replicative senescen~e.~~’ Hybrid fusions between senescent and early-passage (young) HDF demonstrate a dominant inhibitory effect of the senescent cell nucleus on DNA synthesis in the nucleus of the young cell, which can be abolished by chemical agents that block protein and RNA synthesi~.~**,~ Moreover, microinjection of polyA+ RNA derived from senescent HDF into young