• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of narLink to Publisher's site
Nucleic Acids Res. Aug 25, 1995; 23(16): 3244–3251.
PMCID: PMC307184

Cataloging altered gene expression in young and senescent cells using enhanced differential display.

Abstract

Recently, a novel PCR-based technique, differential display (DD), has facilitated the study of differentially expressed genes at the mRNA level. We report here an improved version of DD, which we call Enhanced Differential Display (EDD). We have modified the technique to enhance reproducibility and to facilitate sequencing and cloning. Using EDD, we have generated and verified a catalog of genes that are differentially expressed between young and senescent human diploid fibroblasts (HDF). From 168 genetags that were identified initially, 84 could be sequenced directly from PCR amplified bands. These sequences represent 27 known genes and 37 novel genes. By Northern blot analysis we have confirmed the differential expression of a total of 23 genes (12 known, 11 novel), while 19 (seven known, 12 novel) did not show differential expression. Several of the known genes were previously observed by others to be differentially expressed between young and senescent fibroblasts, thereby validating the technique.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.9M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Liang P, Averboukh L, Keyomarsi K, Sager R, Pardee AB. Differential display and cloning of messenger RNAs from human breast cancer versus mammary epithelial cells. Cancer Res. 1992 Dec 15;52(24):6966–6968. [PubMed]
  • Liang P, Averboukh L, Pardee AB. Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization. Nucleic Acids Res. 1993 Jul 11;21(14):3269–3275. [PMC free article] [PubMed]
  • Nishio Y, Aiello LP, King GL. Glucose induced genes in bovine aortic smooth muscle cells identified by mRNA differential display. FASEB J. 1994 Jan;8(1):103–106. [PubMed]
  • Sun Y, Hegamyer G, Colburn NH. Molecular cloning of five messenger RNAs differentially expressed in preneoplastic or neoplastic JB6 mouse epidermal cells: one is homologous to human tissue inhibitor of metalloproteinases-3. Cancer Res. 1994 Mar 1;54(5):1139–1144. [PubMed]
  • Bauer D, Müller H, Reich J, Riedel H, Ahrenkiel V, Warthoe P, Strauss M. Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR). Nucleic Acids Res. 1993 Sep 11;21(18):4272–4280. [PMC free article] [PubMed]
  • Haag E, Raman V. Effects of primer choice and source of Taq DNA polymerase on the banding patterns of differential display RT-PCR. Biotechniques. 1994 Aug;17(2):226–228. [PubMed]
  • Reeves SA, Rubio MP, Louis DN. General method for PCR amplification and direct sequencing of mRNA differential display products. Biotechniques. 1995 Jan;18(1):18–20. [PubMed]
  • Ito T, Kito K, Adati N, Mitsui Y, Hagiwara H, Sakaki Y. Fluorescent differential display: arbitrarily primed RT-PCR fingerprinting on an automated DNA sequencer. FEBS Lett. 1994 Sep 5;351(2):231–236. [PubMed]
  • Chen Z, Swisshelm K, Sager R. A cautionary note on reaction tubes for differential display and cDNA amplification in thermal cycling. Biotechniques. 1994 Jun;16(6):1002–1006. [PubMed]
  • Lohmann J, Schickle H, Bosch TC. REN display, a rapid and efficient method for nonradioactive differential display and mRNA isolation. Biotechniques. 1995 Feb;18(2):200–202. [PubMed]
  • Li F, Barnathan ES, Karikó K. Rapid method for screening and cloning cDNAs generated in differential mRNA display: application of northern blot for affinity capturing of cDNAs. Nucleic Acids Res. 1994 May 11;22(9):1764–1765. [PMC free article] [PubMed]
  • Mou L, Miller H, Li J, Wang E, Chalifour L. Improvements to the differential display method for gene analysis. Biochem Biophys Res Commun. 1994 Mar 15;199(2):564–569. [PubMed]
  • Callard D, Lescure B, Mazzolini L. A method for the elimination of false positives generated by the mRNA differential display technique. Biotechniques. 1994 Jun;16(6):1096–1103. [PubMed]
  • HAYFLICK L, MOORHEAD PS. The serial cultivation of human diploid cell strains. Exp Cell Res. 1961 Dec;25:585–621. [PubMed]
  • HAYFLICK L. THE LIMITED IN VITRO LIFETIME OF HUMAN DIPLOID CELL STRAINS. Exp Cell Res. 1965 Mar;37:614–636. [PubMed]
  • Hayflick L. Aging under glass. Exp Gerontol. 1970 Dec;5(4):291–303. [PubMed]
  • Martin GM, Sprague CA, Epstein CJ. Replicative life-span of cultivated human cells. Effects of donor's age, tissue, and genotype. Lab Invest. 1970 Jul;23(1):86–92. [PubMed]
  • Bruce SA, Deamond SF, Ts'o PO. In vitro senescence of Syrian hamster mesenchymal cells of fetal to aged adult origin. Inverse relationship between in vivo donor age and in vitro proliferative capacity. Mech Ageing Dev. 1986 Apr;34(2):151–173. [PubMed]
  • Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature. 1990 May 31;345(6274):458–460. [PubMed]
  • Wistrom C, Villeponteau B. Cloning and expression of SAG: a novel marker of cellular senescence. Exp Cell Res. 1992 Apr;199(2):355–362. [PubMed]
  • West MD. The cellular and molecular biology of skin aging. Arch Dermatol. 1994 Jan;130(1):87–95. [PubMed]
  • Seshadri T, Campisi J. Repression of c-fos transcription and an altered genetic program in senescent human fibroblasts. Science. 1990 Jan 12;247(4939):205–209. [PubMed]
  • Irving J, Feng J, Wistrom C, Pikaart M, Villeponteau B. An altered repertoire of fos/jun (AP-1) at the onset of replicative senescence. Exp Cell Res. 1992 Sep;202(1):161–166. [PubMed]
  • Goldstein S, Moerman EJ, Baxter RC. Accumulation of insulin-like growth factor binding protein-3 in conditioned medium of human fibroblasts increases with chronologic age of donor and senescence in vitro. J Cell Physiol. 1993 Aug;156(2):294–302. [PubMed]
  • Goldstein S. Replicative senescence: the human fibroblast comes of age. Science. 1990 Sep 7;249(4973):1129–1133. [PubMed]
  • Doggett DL, Rotenberg MO, Pignolo RJ, Phillips PD, Cristofalo VJ. Differential gene expression between young and senescent, quiescent WI-38 cells. Mech Ageing Dev. 1992 Sep;65(2-3):239–255. [PubMed]
  • Campisi J. Gene expression in quiescent and senescent fibroblasts. Ann N Y Acad Sci. 1992 Nov 21;663:195–201. [PubMed]
  • Cristofalo VJ, Gerhard GS, Pignolo RJ. Molecular biology of aging. Surg Clin North Am. 1994 Feb;74(1):1–21. [PubMed]
  • Pignolo RJ, Rotenberg MO, Cristofalo VJ. Alterations in contact and density-dependent arrest state in senescent WI-38 cells. In Vitro Cell Dev Biol Anim. 1994 Jul;30A(7):471–476. [PubMed]
  • Walton J. The role of limited cell replicative capacity in pathological age change. A review. Mech Ageing Dev. 1982 Jul;19(3):217–244. [PubMed]
  • Satoh Y, Kashimura M, Kaneko S, Karasaki Y, Higashi K, Gotoh S. Cloning of cDNAs with possible association with senescence and immortalization of human cells. Mutat Res. 1994 Feb;316(1):25–36. [PubMed]
  • Sager R, Anisowicz A, Neveu M, Liang P, Sotiropoulou G. Identification by differential display of alpha 6 integrin as a candidate tumor suppressor gene. FASEB J. 1993 Jul;7(10):964–970. [PubMed]
  • Watson MA, Fleming TP. Isolation of differentially expressed sequence tags from human breast cancer. Cancer Res. 1994 Sep 1;54(17):4598–4602. [PubMed]
  • Utans U, Liang P, Wyner LR, Karnovsky MJ, Russell ME. Chronic cardiac rejection: identification of five upregulated genes in transplanted hearts by differential mRNA display. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6463–6467. [PMC free article] [PubMed]
  • Zimmermann JW, Schultz RM. Analysis of gene expression in the preimplantation mouse embryo: use of mRNA differential display. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5456–5460. [PMC free article] [PubMed]
  • Aiello LP, Robinson GS, Lin YW, Nishio Y, King GL. Identification of multiple genes in bovine retinal pericytes altered by exposure to elevated levels of glucose by using mRNA differential display. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6231–6235. [PMC free article] [PubMed]
  • Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. [PubMed]
  • Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967–971. [PubMed]
  • Sheets MD, Ogg SC, Wickens MP. Point mutations in AAUAAA and the poly (A) addition site: effects on the accuracy and efficiency of cleavage and polyadenylation in vitro. Nucleic Acids Res. 1990 Oct 11;18(19):5799–5805. [PMC free article] [PubMed]
  • Goldstein S, Moerman EJ, Fujii S, Sobel BE. Overexpression of plasminogen activator inhibitor type-1 in senescent fibroblasts from normal subjects and those with Werner syndrome. J Cell Physiol. 1994 Dec;161(3):571–579. [PubMed]
  • Fazio MJ, Olsen DR, Kuivaniemi H, Chu ML, Davidson JM, Rosenbloom J, Uitto J. Isolation and characterization of human elastin cDNAs, and age-associated variation in elastin gene expression in cultured skin fibroblasts. Lab Invest. 1988 Mar;58(3):270–277. [PubMed]
  • Pignolo RJ, Cristofalo VJ, Rotenberg MO. Senescent WI-38 cells fail to express EPC-1, a gene induced in young cells upon entry into the G0 state. J Biol Chem. 1993 Apr 25;268(12):8949–8957. [PubMed]
  • West MD, Pereira-Smith OM, Smith JR. Replicative senescence of human skin fibroblasts correlates with a loss of regulation and overexpression of collagenase activity. Exp Cell Res. 1989 Sep;184(1):138–147. [PubMed]
  • Shay JW, West MD, Wright WE. Re-expression of senescent markers in deinduced reversibly immortalized cells. Exp Gerontol. 1992 Sep-Dec;27(5-6):477–492. [PubMed]
  • Kumar S, Vinci JM, Millis AJ, Baglioni C. Expression of interleukin-1 alpha and beta in early passage fibroblasts from aging individuals. Exp Gerontol. 1993 Nov-Dec;28(6):505–513. [PubMed]
  • Dumas M, Chaudagne C, Bonté F, Meybeck A. In vitro biosynthesis of type I and III collagens by human dermal fibroblasts from donors of increasing age. Mech Ageing Dev. 1994 Mar;73(3):179–187. [PubMed]
  • Furth JJ. The steady-state levels of type I collagen mRNA are reduced in senescent fibroblasts. J Gerontol. 1991 May;46(3):B122–B124. [PubMed]
  • Wong GH, Goeddel DV. Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science. 1988 Nov 11;242(4880):941–944. [PubMed]
  • Kumar S, Millis AJ, Baglioni C. Expression of interleukin 1-inducible genes and production of interleukin 1 by aging human fibroblasts. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4683–4687. [PMC free article] [PubMed]
  • Kiefer MC, Masiarz FR, Bauer DM, Zapf J. Identification and molecular cloning of two new 30-kDa insulin-like growth factor binding proteins isolated from adult human serum. J Biol Chem. 1991 May 15;266(14):9043–9049. [PubMed]
  • Grigoriev VG, Moerman EJ, Goldstein S. Senescence and cell density of human diploid fibroblasts influence metabolism of insulin-like growth factor binding proteins. J Cell Physiol. 1994 Jul;160(1):203–211. [PubMed]
  • Moerman EJ, Thweatt R, Moerman AM, Jones RA, Goldstein S. Insulin-like growth factor binding protein-3 is overexpressed in senescent and quiescent human fibroblasts. Exp Gerontol. 1993 Jul-Oct;28(4-5):361–370. [PubMed]
  • Eleftheriou CS, Trakas N, Kokla A, Tzartos SJ. A group of three fibroblast secreted polypeptides suppressed by cellular ageing and interferon-gamma. Biochim Biophys Acta. 1993 Jan 22;1180(3):304–312. [PubMed]
  • Scarpati EM, Wen D, Broze GJ, Jr, Miletich JP, Flandermeyer RR, Siegel NR, Sadler JE. Human tissue factor: cDNA sequence and chromosome localization of the gene. Biochemistry. 1987 Aug 25;26(17):5234–5238. [PubMed]
  • Harn HJ, Isola N, Cooper DL. The multispecific cell adhesion molecule CD44 is represented in reticulocyte cDNA. Biochem Biophys Res Commun. 1991 Aug 15;178(3):1127–1134. [PubMed]
  • Holmes WE, Sliwkowski MX, Akita RW, Henzel WJ, Lee J, Park JW, Yansura D, Abadi N, Raab H, Lewis GD, et al. Identification of heregulin, a specific activator of p185erbB2. Science. 1992 May 22;256(5060):1205–1210. [PubMed]
  • Hsu LC, Tani K, Fujiyoshi T, Kurachi K, Yoshida A. Cloning of cDNAs for human aldehyde dehydrogenases 1 and 2. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3771–3775. [PMC free article] [PubMed]
  • Sakai K, Hirai M, Kudoh J, Minoshima S, Shimizu N. Molecular cloning and chromosomal mapping of a cDNA encoding human 80K-L protein: major substrate for protein kinase C. Genomics. 1992 Sep;14(1):175–178. [PubMed]
  • Richards AJ, al-Imara L, Carter NP, Lloyd JC, Leversha MA, Pope FM. Localization of the gene (LAMA4) to chromosome 6q21 and isolation of a partial cDNA encoding a variant laminin A chain. Genomics. 1994 Jul 1;22(1):237–239. [PubMed]
  • Zeng G, Millis AJ. Expression of 72-kDa gelatinase and TIMP-2 in early and late passage human fibroblasts. Exp Cell Res. 1994 Jul;213(1):148–155. [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...