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. 1997 Oct 1;25(19):3823–3831. doi: 10.1093/nar/25.19.3823

Identification of rapid turnover transcripts overexpressed in thyroid tumors and thyroid cancer cell lines: use of a targeted differential RNA display method to select for mRNA subsets.

R Gonsky 1, J A Knauf 1, R Elisei 1, J W Wang 1, S Su 1, J A Fagin 1
PMCID: PMC146961  PMID: 9380504

Abstract

The mRNAs of transiently expressed proteins such as cytokines and proto-oncogenes are commonly subject to rapid transcriptional activation and degradation. Transcript turnover is determined in part by association of certain proteins with consensus AU-rich motifs (AUUUA) in the 3'-untranslated region of the transcripts. Here we report a modification of differential RNA display (DRD) to detect differentially expressed rapid turnover mRNAs containing AU-rich motifs from thyroid cancer tissues and cell lines. RNA of normal and thyroid cancer tissues was differentially displayed using a 3'anchor primer to the poly(A) tail and an arbitrary 5'primer incorporating an AUUUA sequence. The appropriateness of the strategy was established by its ability to display known early response genes, such as c- fos, using partially degenerate primers. To test whether the novel cDNAs isolated coded for transcripts subject to rapid turnover, they were used as probes for Northern blots of RNA from clonal human thyroid carcinoma cell lines treated for varying periods with either cycloheximide or actinomycin D. A number of novel differentially expressed cDNA fragments were isolated from human papillary thyroid carcinoma tissues, among them a cDNA with zinc finger motifs and homology to other zinc finger proteins. Using this fragment to probe a cDNA library, a full-length cDNA (ZnF20) was isolated that was 4333 bp in length and contained an open reading frame of 1029 amino acids. The ZnF20 cDNA hybridized to multiple transcripts in a thyroid cancer cell line (8.0, 4.5 and 2 kb) that increased after cycloheximide treatment and decayed <2 h after addition of actinomycin D. The ZnF20 mRNA was overexpressed in three of six thyroid papillary carcinomas as compared with paired normal thyroid tissue controls. The data presented here support the use of a targeted DRD approach for the isolation of rapid turnover mRNAs, many of which may be interesting candidate oncogenes.

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Selected References

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  1. Alberta J. A., Rundell K., Stiles C. D. Identification of an activity that interacts with the 3'-untranslated region of c-myc mRNA and the role of its target sequence in mediating rapid mRNA degradation. J Biol Chem. 1994 Feb 11;269(6):4532–4538. [PubMed] [Google Scholar]
  2. Alitalo K., Saksela K., Winqvist R., Alitalo R., Keski-Oja J., Laiho M., Ilvonen M., Knuutila S., de la Chapelle A. Acute myelogenous leukaemia with c-myc amplification and double minute chromosomes. Lancet. 1985 Nov 9;2(8463):1035–1039. doi: 10.1016/s0140-6736(85)90907-9. [DOI] [PubMed] [Google Scholar]
  3. Arnold A., Kim H. G., Gaz R. D., Eddy R. L., Fukushima Y., Byers M. G., Shows T. B., Kronenberg H. M. Molecular cloning and chromosomal mapping of DNA rearranged with the parathyroid hormone gene in a parathyroid adenoma. J Clin Invest. 1989 Jun;83(6):2034–2040. doi: 10.1172/JCI114114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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. doi: 10.1093/nar/21.18.4272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bergerheim U., Nordenskjöld M., Collins V. P. Deletion mapping in human renal cell carcinoma. Cancer Res. 1989 Mar 15;49(6):1390–1396. [PubMed] [Google Scholar]
  6. Bohjanen P. R., Petryniak B., June C. H., Thompson C. B., Lindsten T. AU RNA-binding factors differ in their binding specificities and affinities. J Biol Chem. 1992 Mar 25;267(9):6302–6309. [PubMed] [Google Scholar]
  7. Bohjanen P. R., Petryniak B., June C. H., Thompson C. B., Lindsten T. An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3' untranslated region of lymphokine mRNA. Mol Cell Biol. 1991 Jun;11(6):3288–3295. doi: 10.1128/mcb.11.6.3288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brauch H., Johnson B., Hovis J., Yano T., Gazdar A., Pettengill O. S., Graziano S., Sorenson G. D., Poiesz B. J., Minna J. Molecular analysis of the short arm of chromosome 3 in small-cell and non-small-cell carcinoma of the lung. N Engl J Med. 1987 Oct 29;317(18):1109–1113. doi: 10.1056/NEJM198710293171803. [DOI] [PubMed] [Google Scholar]
  9. Brodeur G. M., Seeger R. C., Schwab M., Varmus H. E., Bishop J. M. Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science. 1984 Jun 8;224(4653):1121–1124. doi: 10.1126/science.6719137. [DOI] [PubMed] [Google Scholar]
  10. Calabrò V., Pengue G., Bartoli P. C., Pagliuca A., Featherstone T., Lania L. Positional cloning of cDNAs from the human chromosome 3p21-22 region identifies a clustered organization of zinc-finger genes. Hum Genet. 1995 Jan;95(1):18–21. doi: 10.1007/BF00225067. [DOI] [PubMed] [Google Scholar]
  11. Caput D., Beutler B., Hartog K., Thayer R., Brown-Shimer S., Cerami A. Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1670–1674. doi: 10.1073/pnas.83.6.1670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Carroll P. R., Murty V. V., Reuter V., Jhanwar S., Fair W. R., Whitmore W. F., Chaganti R. S. Abnormalities at chromosome region 3p12-14 characterize clear cell renal carcinoma. Cancer Genet Cytogenet. 1987 Jun;26(2):253–259. doi: 10.1016/0165-4608(87)90059-8. [DOI] [PubMed] [Google Scholar]
  13. Chen J., Heerdt B. G., Augenlicht L. H. Presence and instability of repetitive elements in sequences the altered expression of which characterizes risk for colonic cancer. Cancer Res. 1995 Jan 1;55(1):174–180. [PubMed] [Google Scholar]
  14. Chomczynski P., Mackey K. One-hour downward capillary blotting of RNA at neutral pH. Anal Biochem. 1994 Sep;221(2):303–305. doi: 10.1006/abio.1994.1416. [DOI] [PubMed] [Google Scholar]
  15. Daly M. C., Douglas J. B., Bleehen N. M., Hastleton P., Twentyman P. R., Sundaresan V., Carritt B., Bergh J., Rabbitts P. H. An unusually proximal deletion on the short arm of chromosome 3 in a patient with small cell lung cancer. Genomics. 1991 Jan;9(1):113–119. doi: 10.1016/0888-7543(91)90227-6. [DOI] [PubMed] [Google Scholar]
  16. Devilee P., van den Broek M., Kuipers-Dijkshoorn N., Kolluri R., Khan P. M., Pearson P. L., Cornelisse C. J. At least four different chromosomal regions are involved in loss of heterozygosity in human breast carcinoma. Genomics. 1989 Oct;5(3):554–560. doi: 10.1016/0888-7543(89)90023-2. [DOI] [PubMed] [Google Scholar]
  17. Ehlen T., Dubeau L. Loss of heterozygosity on chromosomal segments 3p, 6q and 11p in human ovarian carcinomas. Oncogene. 1990 Feb;5(2):219–223. [PubMed] [Google Scholar]
  18. Fort P., Rech J., Vie A., Piechaczyk M., Bonnieu A., Jeanteur P., Blanchard J. M. Regulation of c-fos gene expression in hamster fibroblasts: initiation and elongation of transcription and mRNA degradation. Nucleic Acids Res. 1987 Jul 24;15(14):5657–5667. doi: 10.1093/nar/15.14.5657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ganly P. S., Jarad N., Rudd R. M., Rabbitts P. H. PCR-based RFLP analysis allows genotyping of the short arm of chromosome 3 in small biopsies from patients with lung cancer. Genomics. 1992 Feb;12(2):221–228. doi: 10.1016/0888-7543(92)90369-4. [DOI] [PubMed] [Google Scholar]
  20. Giancotti V., Buratti E., Perissin L., Zorzet S., Balmain A., Portella G., Fusco A., Goodwin G. H. Analysis of the HMGI nuclear proteins in mouse neoplastic cells induced by different procedures. Exp Cell Res. 1989 Oct;184(2):538–545. doi: 10.1016/0014-4827(89)90352-2. [DOI] [PubMed] [Google Scholar]
  21. Glaichenhaus N., Cuzin F. A role for ID repetitive sequences in growth- and transformation-dependent regulation of gene expression in rat fibroblasts. Cell. 1987 Sep 25;50(7):1081–1089. doi: 10.1016/0092-8674(87)90174-7. [DOI] [PubMed] [Google Scholar]
  22. Harland R., Misher L. Stability of RNA in developing Xenopus embryos and identification of a destabilizing sequence in TFIIIA messenger RNA. Development. 1988 Apr;102(4):837–852. doi: 10.1242/dev.102.4.837. [DOI] [PubMed] [Google Scholar]
  23. Herrmann M. A., Hay I. D., Bartelt D. H., Jr, Ritland S. R., Dahl R. J., Grant C. S., Jenkins R. B. Cytogenetic and molecular genetic studies of follicular and papillary thyroid cancers. J Clin Invest. 1991 Nov;88(5):1596–1604. doi: 10.1172/JCI115472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jackson R. J. Cytoplasmic regulation of mRNA function: the importance of the 3' untranslated region. Cell. 1993 Jul 16;74(1):9–14. doi: 10.1016/0092-8674(93)90290-7. [DOI] [PubMed] [Google Scholar]
  25. Knox W. E., Tremblay G. C., Spanier B. B., Friedell G. H. Glutaminase activities in normal and neoplastic tissues of the rat. Cancer Res. 1967 Aug;27(8):1456–1458. [PubMed] [Google Scholar]
  26. Kovacs G., Erlandsson R., Boldog F., Ingvarsson S., Müller-Brechlin R., Klein G., Sümegi J. Consistent chromosome 3p deletion and loss of heterozygosity in renal cell carcinoma. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1571–1575. doi: 10.1073/pnas.85.5.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lammie G. A., Fantl V., Smith R., Schuuring E., Brookes S., Michalides R., Dickson C., Arnold A., Peters G. D11S287, a putative oncogene on chromosome 11q13, is amplified and expressed in squamous cell and mammary carcinomas and linked to BCL-1. Oncogene. 1991 Mar;6(3):439–444. [PubMed] [Google Scholar]
  28. Lebwohl D. E., Muise-Helmericks R., Sepp-Lorenzino L., Serve S., Timaul M., Bol R., Borgen P., Rosen N. A truncated cyclin D1 gene encodes a stable mRNA in a human breast cancer cell line. Oncogene. 1994 Jul;9(7):1925–1929. [PubMed] [Google Scholar]
  29. Liang P., Pardee A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967–971. doi: 10.1126/science.1354393. [DOI] [PubMed] [Google Scholar]
  30. Lin C. C., Alitalo K., Schwab M., George D., Varmus H. E., Bishop J. M. Evolution of karyotypic abnormalities and C-MYC oncogene amplification in human colonic carcinoma cell lines. Chromosoma. 1985;92(1):11–15. doi: 10.1007/BF00327240. [DOI] [PubMed] [Google Scholar]
  31. Lothe R. A., Fosså S. D., Stenwig A. E., Nakamura Y., White R., Børresen A. L., Brøgger A. Loss of 3p or 11p alleles is associated with testicular cancer tumors. Genomics. 1989 Jul;5(1):134–138. doi: 10.1016/0888-7543(89)90097-9. [DOI] [PubMed] [Google Scholar]
  32. Matsuno T., Hirai H. Glutamine synthetase and glutaminase activities in various hepatoma cells. Biochem Int. 1989 Aug;19(2):219–225. [PubMed] [Google Scholar]
  33. Medina M. A., Sánchez-Jiménez F., Márquez J., Rodríguez Quesada A., Núez de Castro I. Relevance of glutamine metabolism to tumor cell growth. Mol Cell Biochem. 1992 Jul 6;113(1):1–15. doi: 10.1007/BF00230880. [DOI] [PubMed] [Google Scholar]
  34. Murphy D., Brickell P. M., Latchman D. S., Willison K., Rigby P. W. Transcripts regulated during normal embryonic development and oncogenic transformation share a repetitive element. Cell. 1983 Dec;35(3 Pt 2):865–871. doi: 10.1016/0092-8674(83)90119-8. [DOI] [PubMed] [Google Scholar]
  35. Naylor S. L., Johnson B. E., Minna J. D., Sakaguchi A. Y. Loss of heterozygosity of chromosome 3p markers in small-cell lung cancer. Nature. 1987 Oct 1;329(6138):451–454. doi: 10.1038/329451a0. [DOI] [PubMed] [Google Scholar]
  36. Okubo K., Hori N., Matoba R., Niiyama T., Fukushima A., Kojima Y., Matsubara K. Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression. Nat Genet. 1992 Nov;2(3):173–179. doi: 10.1038/ng1192-173. [DOI] [PubMed] [Google Scholar]
  37. Peltz S. W., Jacobson A. mRNA stability: in trans-it. Curr Opin Cell Biol. 1992 Dec;4(6):979–983. doi: 10.1016/0955-0674(92)90129-z. [DOI] [PubMed] [Google Scholar]
  38. Pengue G., Calabrò V., Cannada-Bartoli P., De Luca P., Esposito T., Taillon-Miller P., LaForgia S., Druck T., Huebner K., D'Urso M. YAC-assisted cloning of transcribed sequences from the human chromosome 3p21 region. Hum Mol Genet. 1993 Jun;2(6):791–796. doi: 10.1093/hmg/2.6.791. [DOI] [PubMed] [Google Scholar]
  39. Popescu N. C., Chahinian A. P., DiPaolo J. A. Nonrandom chromosome alterations in human malignant mesothelioma. Cancer Res. 1988 Jan 1;48(1):142–147. [PubMed] [Google Scholar]
  40. Rahmsdorf H. J., Schönthal A., Angel P., Litfin M., Rüther U., Herrlich P. Posttranscriptional regulation of c-fos mRNA expression. Nucleic Acids Res. 1987 Feb 25;15(4):1643–1659. doi: 10.1093/nar/15.4.1643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ralph D., McClelland M., Welsh J. RNA fingerprinting using arbitrarily primed PCR identifies differentially regulated RNAs in mink lung (Mv1Lu) cells growth arrested by transforming growth factor beta 1. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10710–10714. doi: 10.1073/pnas.90.22.10710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sachs A. B. Messenger RNA degradation in eukaryotes. Cell. 1993 Aug 13;74(3):413–421. doi: 10.1016/0092-8674(93)80043-e. [DOI] [PubMed] [Google Scholar]
  43. Sasi R., Hoo J. J., Samuel I. P., Tainaka T., Shiferaw S., Lin C. C. Chromosome aberrations and oncogene alterations in two new breast tumor cell lines. Cancer Genet Cytogenet. 1991 Feb;51(2):239–254. doi: 10.1016/0165-4608(91)90137-j. [DOI] [PubMed] [Google Scholar]
  44. Shaw G., Kamen R. A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell. 1986 Aug 29;46(5):659–667. doi: 10.1016/0092-8674(86)90341-7. [DOI] [PubMed] [Google Scholar]
  45. Shay J. W., Baba T., Zhan Q. M., Kamimura N., Cuthbert J. A. HeLaTG cells have mitochondrial DNA inserted into the c-myc oncogene. Oncogene. 1991 Oct;6(10):1869–1874. [PubMed] [Google Scholar]
  46. Shibuya M., Yokota J., Ueyama Y. Amplification and expression of a cellular oncogene (c-myc) in human gastric adenocarcinoma cells. Mol Cell Biol. 1985 Feb;5(2):414–418. doi: 10.1128/mcb.5.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Singh K., Carey M., Saragosti S., Botchan M. Expression of enhanced levels of small RNA polymerase III transcripts encoded by the B2 repeats in simian virus 40-transformed mouse cells. Nature. 1985 Apr 11;314(6011):553–556. doi: 10.1038/314553a0. [DOI] [PubMed] [Google Scholar]
  48. Stone B., Wharton W. Targeted RNA fingerprinting: the cloning of differentially-expressed cDNA fragments enriched for members of the zinc finger gene family. Nucleic Acids Res. 1994 Jul 11;22(13):2612–2618. doi: 10.1093/nar/22.13.2612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Südhof T. C., Goldstein J. L., Brown M. S., Russell D. W. The LDL receptor gene: a mosaic of exons shared with different proteins. Science. 1985 May 17;228(4701):815–822. doi: 10.1126/science.2988123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Terrier P., Sheng Z. M., Schlumberger M., Tubiana M., Caillou B., Travagli J. P., Fragu P., Parmentier C., Riou G. Structure and expression of c-myc and c-fos proto-oncogenes in thyroid carcinomas. Br J Cancer. 1988 Jan;57(1):43–47. doi: 10.1038/bjc.1988.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tsujimoto Y., Yunis J., Onorato-Showe L., Erikson J., Nowell P. C., Croce C. M. Molecular cloning of the chromosomal breakpoint of B-cell lymphomas and leukemias with the t(11;14) chromosome translocation. Science. 1984 Jun 29;224(4656):1403–1406. doi: 10.1126/science.6610211. [DOI] [PubMed] [Google Scholar]
  52. Welsh J., Chada K., Dalal S. S., Cheng R., Ralph D., McClelland M. Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 1992 Oct 11;20(19):4965–4970. doi: 10.1093/nar/20.19.4965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Whang-Peng J., Kao-Shan C. S., Lee E. C., Bunn P. A., Carney D. N., Gazdar A. F., Minna J. D. Specific chromosome defect associated with human small-cell lung cancer; deletion 3p(14-23). Science. 1982 Jan 8;215(4529):181–182. doi: 10.1126/science.6274023. [DOI] [PubMed] [Google Scholar]
  54. Winqvist R., Knuutila S., Leprince D., Stehelin D., Alitalo K. Mapping of amplified c-myb oncogene, sister chromatid exchanges, and karyotypic analysis of the COLO 205 colon carcinoma cell line. Cancer Genet Cytogenet. 1985 Nov;18(3):251–264. doi: 10.1016/0165-4608(85)90090-1. [DOI] [PubMed] [Google Scholar]
  55. Yokota J., Tsukada Y., Nakajima T., Gotoh M., Shimosato Y., Mori N., Tsunokawa Y., Sugimura T., Terada M. Loss of heterozygosity on the short arm of chromosome 3 in carcinoma of the uterine cervix. Cancer Res. 1989 Jul 1;49(13):3598–3601. [PubMed] [Google Scholar]
  56. Zeki K., Spambalg D., Sharifi N., Gonsky R., Fagin J. A. Mutations of the adenomatous polyposis coli gene in sporadic thyroid neoplasms. J Clin Endocrinol Metab. 1994 Nov;79(5):1317–1321. doi: 10.1210/jcem.79.5.7962323. [DOI] [PubMed] [Google Scholar]

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