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

Genesthat play arole inthesenescent arrest ofcellular replication arelikely tobeoverexpressed inhuman diploid fibroblasts (HDF)derived fromsubjects withWernersyndrome (WS)because these cellshaveaseverely curtailed replicative life span.Toidentify some ofthese genes,acDNAlibrary was constructed fromWS HDF after theyhadbeenserum depleted andrepleted (5daysinmediumcontaining 1% serum followed by24hin mediumcontaining 20%serum). Differential screening of7,500colonies revealed 102clones thathybridized preferentially with[32PJcDNA derived fromRNA ofWS cells compared with[32PJcDNA derived fromnormal HDF.Cross-hybridization andpartial DNA sequencedetermination identified 18independent gene sequences, 9ofthemknownand9unknown. Theknowngenesincluded oal(I) procollagen,a2(I) procollagen, fibronectin, ferritin heavychain, insulinlike growthfactor-binding protein-3 (IGFBP-3), osteonectin, humantissue plasminogen activator inhibitor type1,thrombospondin, andaB-crystallin. Thenineunknownclones included twonovel genesequencesandsevenadditional sequencesthatcontained bothnovel segments andtheAluclass ofrepetitive shortinterspersed nuclear elements; five ofthese sevenAlu+clones alsocontained thelong interspersed nuclear element 1 (KpnI) family ofrepetitive elements. Northern (RNA)analysis, usingthe18 sequencesasprobes, showedhigher levels ofthese mRNAsinWS HDF thaninnormalHDF.Fiveselected mRNAsstudied ingreater detail [al(I) procollagen, fibronectin, insulinlike growthfactor-binding protein-3, WS3-10, andWS9-14] showedhigher mRNA levels inbothWS andlate-passage normalHDF thaninearlypassagenormalHDFatvarious intervals following serum depletion/repletion andafter subculture andgrowth fromsparsetohigh-density confluent arrest. Theseresults indicate that senescenceofbothWS andnormal HDFisaccompanied byoverexpression ofsimilar setsofdiverse geneswhich may play a role inthesenescent arrest ofcellular replication andinthegenesis ofWS,normalbiological aging, andattendant diseases. Biological aging, aninevitable process inmulticellular organisms, features twosetsofinterdependent events: functional decline andanexponential rise intheincidence of degenerative andneoplastic diseases (27). Themechanism of aging remains unknown, butitseemsclear that theprogressivelossofcellular replicative capacity inmanytissues is inextricably involved (26, 27,51,55). Humandiploid fibroblasts (HDF)havealimited replicative life spanandprovide avaluable modelforstudies ofreplicative senescence (14, 36). Earlier studies havepursued theerrorcatastrophe and genetic damagehypotheses aspossible mechanisms, but little evidence hasbeenmarshalled tosupport these ideas. Ontheother hand, theconcept ofagenetically programmed, quasidifferentiative process hasincreasingly beeninvoked forseveral reasons (28). First, senescence ofHDF islinked tothenumberoftimes thecell population doubles uptoa critical limit rather thantothemetabolic orcalendar time (see reference 28andreferences therein). Second, cell fusion experiments involving heterokaryons, hybrid cells containinga late-passage senescent (old) nucleus andanearlypassage proliferation competent (young) nucleus inasingle cytoplasm, clearly demonstrate thattheoldcellhasa dominant inhibitory effect onnuclear DNA synthesis inthe youngcell. Additionally, this inhibition canbeabolished by