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. 1998 Feb 15;26(4):1116–1123. doi: 10.1093/nar/26.4.1116

Differential screening and suppression subtractive hybridization identified genes differentially expressed in an estrogen receptor-positive breast carcinoma cell line.

W W Kuang 1, D A Thompson 1, R V Hoch 1, R J Weigel 1
PMCID: PMC147366  PMID: 9461476

Abstract

Differences in gene expression are likely to explain the phenotypic differences between hormone-responsive and hormone-unresponsive breast cancer. We have identified differentially expressed cDNAs in the estrogen receptor (ER)-positive MCF7 breast carcinoma cell line compared with the ER-negative MDA-MB-231 breast carcinoma cell line. Differential screening isolated four differentially expressed genes: cytokeratin 8, cytokeratin 18, Hsp27 and GPCR -Br. To identify differentially expressed genes of lower abundance, suppression subtractive hybridization was utilized and 29 differentially expressed clones were isolated. Sequence analysis revealed that 11 clones were from previously described genes: HEK8, neuropeptide Y receptor Y1, p21 WAF-1, p55 PIK, cytokeratin 18 (cloned twice), fructose-1,6-biphosphatase, cytokeratin 8, TGFbeta1 binding protein, elongation factor 1alpha2 and pS2. The remaining 18 clones did not match sequences in the GenBank/EMBL database, indicating that they may be novel genes. Expression of pS2, neuropeptide Y receptor Y1 and three novel clones was induced by estradiol, indicating estrogen-responsiveness. The expression pattern of one novel gene, DEME -6, correlated with expression of ER and ERF -1/ AP -2gamma in a panel of breast carcinoma cell lines. A 2.6 kb cDNA of DEME -6 was sequenced and contains an open reading frame of 574 amino acids that demonstrates 62.4% similarity with a gene from Caenorhabditis elegans chromosome III. Expression of DEME -6 was also detected in primary breast carcinomas but not in normal breast tissue, as determined by RT-PCR. These findings support the hypothesis that a set of genes coordinately regulated with ER , but not necessarily estradiol-responsive, are characteristic of the hormone-responsive breast cancer phenotype.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams M. D., Kerlavage A. R., Fleischmann R. D., Fuldner R. A., Bult C. J., Lee N. H., Kirkness E. F., Weinstock K. G., Gocayne J. D., White O. Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nature. 1995 Sep 28;377(6547 Suppl):3–174. [PubMed] [Google Scholar]
  2. Bairoch A., Bucher P., Hofmann K. The PROSITE database, its status in 1997. Nucleic Acids Res. 1997 Jan 1;25(1):217–221. doi: 10.1093/nar/25.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bandziulis R. J., Swanson M. S., Dreyfuss G. RNA-binding proteins as developmental regulators. Genes Dev. 1989 Apr;3(4):431–437. doi: 10.1101/gad.3.4.431. [DOI] [PubMed] [Google Scholar]
  4. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  5. Bosher J. M., Totty N. F., Hsuan J. J., Williams T., Hurst H. C. A family of AP-2 proteins regulates c-erbB-2 expression in mammary carcinoma. Oncogene. 1996 Oct 17;13(8):1701–1707. [PubMed] [Google Scholar]
  6. Brown A. M., Jeltsch J. M., Roberts M., Chambon P. Activation of pS2 gene transcription is a primary response to estrogen in the human breast cancer cell line MCF-7. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6344–6348. doi: 10.1073/pnas.81.20.6344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  8. Carmeci C., Thompson D. A., Ring H. Z., Francke U., Weigel R. J. Identification of a gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer. Genomics. 1997 Nov 1;45(3):607–617. doi: 10.1006/geno.1997.4972. [DOI] [PubMed] [Google Scholar]
  9. Carmeci C., deConinck E. C., Lawton T., Bloch D. A., Weigel R. J. Analysis of estrogen receptor messenger RNA in breast carcinomas from archival specimens is predictive of tumor biology. Am J Pathol. 1997 May;150(5):1563–1570. [PMC free article] [PubMed] [Google Scholar]
  10. Castles C. G., Fuqua S. A., Klotz D. M., Hill S. M. Expression of a constitutively active estrogen receptor variant in the estrogen receptor-negative BT-20 human breast cancer cell line. Cancer Res. 1993 Dec 15;53(24):5934–5939. [PubMed] [Google Scholar]
  11. Diatchenko L., Lau Y. F., Campbell A. P., Chenchik A., Moqadam F., Huang B., Lukyanov S., Lukyanov K., Gurskaya N., Sverdlov E. D. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):6025–6030. doi: 10.1073/pnas.93.12.6025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Edwards D. P., Chamness G. C., McGuire W. L. Estrogen and progesterone receptor proteins in breast cancer. Biochim Biophys Acta. 1979 Dec 19;560(4):457–486. doi: 10.1016/0304-419x(79)90013-1. [DOI] [PubMed] [Google Scholar]
  13. Fisher B., Redmond C., Fisher E. R., Caplan R. Relative worth of estrogen or progesterone receptor and pathologic characteristics of differentiation as indicators of prognosis in node negative breast cancer patients: findings from National Surgical Adjuvant Breast and Bowel Project Protocol B-06. J Clin Oncol. 1988 Jul;6(7):1076–1087. doi: 10.1200/JCO.1988.6.7.1076. [DOI] [PubMed] [Google Scholar]
  14. Fuqua S. A., Fitzgerald S. D., Allred D. C., Elledge R. M., Nawaz Z., McDonnell D. P., O'Malley B. W., Greene G. L., McGuire W. L. Inhibition of estrogen receptor action by a naturally occurring variant in human breast tumors. Cancer Res. 1992 Jan 15;52(2):483–486. [PubMed] [Google Scholar]
  15. Fuqua S. A., Fitzgerald S. D., Chamness G. C., Tandon A. K., McDonnell D. P., Nawaz Z., O'Malley B. W., McGuire W. L. Variant human breast tumor estrogen receptor with constitutive transcriptional activity. Cancer Res. 1991 Jan 1;51(1):105–109. [PubMed] [Google Scholar]
  16. Grandien K. Determination of transcription start sites in the human estrogen receptor gene and identification of a novel, tissue-specific, estrogen receptor-mRNA isoform. Mol Cell Endocrinol. 1996 Feb 5;116(2):207–212. doi: 10.1016/0303-7207(95)03716-0. [DOI] [PubMed] [Google Scholar]
  17. Henry J. A., Nicholson S., Farndon J. R., Westley B. R., May F. E. Measurement of oestrogen receptor mRNA levels in human breast tumours. Br J Cancer. 1988 Nov;58(5):600–605. doi: 10.1038/bjc.1988.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Higgins D. G., Bleasby A. J., Fuchs R. CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci. 1992 Apr;8(2):189–191. doi: 10.1093/bioinformatics/8.2.189. [DOI] [PubMed] [Google Scholar]
  19. Hill S. M., Fuqua S. A., Chamness G. C., Greene G. L., McGuire W. L. Estrogen receptor expression in human breast cancer associated with an estrogen receptor gene restriction fragment length polymorphism. Cancer Res. 1989 Jan 1;49(1):145–148. [PubMed] [Google Scholar]
  20. Horvath T. L., Shanabrough M., Naftolin F., Leranth C. Neuropeptide-Y innervation of estrogen-induced progesterone receptor-containing dopamine cells in the monkey hypothalamus: a triple labeling light and electron microscopic study. Endocrinology. 1993 Jul;133(1):405–414. doi: 10.1210/endo.133.1.8100520. [DOI] [PubMed] [Google Scholar]
  21. Hubank M., Schatz D. G. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res. 1994 Dec 25;22(25):5640–5648. doi: 10.1093/nar/22.25.5640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jiang S. Y., Jordan V. C. Growth regulation of estrogen receptor-negative breast cancer cells transfected with complementary DNAs for estrogen receptor. J Natl Cancer Inst. 1992 Apr 15;84(8):580–591. doi: 10.1093/jnci/84.8.580. [DOI] [PubMed] [Google Scholar]
  23. Kalra S. P., Fuentes M., Fournier A., Parker S. L., Crowley W. R. Involvement of the Y-1 receptor subtype in the regulation of luteinizing hormone secretion by neuropeptide Y in rats. Endocrinology. 1992 Jun;130(6):3323–3330. doi: 10.1210/endo.130.6.1317781. [DOI] [PubMed] [Google Scholar]
  24. Keaveney M., Klug J., Dawson M. T., Nestor P. V., Neilan J. G., Forde R. C., Gannon F. Evidence for a previously unidentified upstream exon in the human oestrogen receptor gene. J Mol Endocrinol. 1991 Feb;6(1):111–115. doi: 10.1677/jme.0.0060111. [DOI] [PubMed] [Google Scholar]
  25. Knight W. A., Livingston R. B., Gregory E. J., McGuire W. L. Estrogen receptor as an independent prognostic factor for early recurrence in breast cancer. Cancer Res. 1977 Dec;37(12):4669–4671. [PubMed] [Google Scholar]
  26. Marck C. 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988 Mar 11;16(5):1829–1836. doi: 10.1093/nar/16.5.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maser R. L., Calvet J. P. Analysis of differential gene expression in the kidney by differential cDNA screening, subtractive cloning, and mRNA differential display. Semin Nephrol. 1995 Jan;15(1):29–42. [PubMed] [Google Scholar]
  28. Masiakowski P., Breathnach R., Bloch J., Gannon F., Krust A., Chambon P. Cloning of cDNA sequences of hormone-regulated genes from the MCF-7 human breast cancer cell line. Nucleic Acids Res. 1982 Dec 20;10(24):7895–7903. doi: 10.1093/nar/10.24.7895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. May F. E., Johnson M. D., Wiseman L. R., Wakeling A. E., Kastner P., Westley B. R. Regulation of progesterone receptor mRNA by oestradiol and antioestrogens in breast cancer cell lines. J Steroid Biochem. 1989 Dec;33(6):1035–1041. doi: 10.1016/0022-4731(89)90406-8. [DOI] [PubMed] [Google Scholar]
  30. McPherson L. A., Baichwal V. R., Weigel R. J. Identification of ERF-1 as a member of the AP2 transcription factor family. Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4342–4347. doi: 10.1073/pnas.94.9.4342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moretti-Rojas I., Fuqua S. A., Montgomery R. A., 3rd, McGuire W. L. A cDNA for the estradiol-regulated 24K protein: control of mRNA levels in MCF-7 cells. Breast Cancer Res Treat. 1988 May;11(2):155–163. doi: 10.1007/BF01805839. [DOI] [PubMed] [Google Scholar]
  32. Nembrot M., Quintana B., Mordoh J. Estrogen receptor gene amplification is found in some estrogen receptor-positive human breast tumors. Biochem Biophys Res Commun. 1990 Jan 30;166(2):601–607. doi: 10.1016/0006-291x(90)90851-d. [DOI] [PubMed] [Google Scholar]
  33. Osborne C. K., Yochmowitz M. G., Knight W. A., 3rd, McGuire W. L. The value of estrogen and progesterone receptors in the treatment of breast cancer. Cancer. 1980 Dec 15;46(12 Suppl):2884–2888. doi: 10.1002/1097-0142(19801215)46:12+<2884::aid-cncr2820461429>3.0.co;2-u. [DOI] [PubMed] [Google Scholar]
  34. Ottaviano Y. L., Issa J. P., Parl F. F., Smith H. S., Baylin S. B., Davidson N. E. Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells. Cancer Res. 1994 May 15;54(10):2552–2555. [PubMed] [Google Scholar]
  35. Pfeffer U. Estrogen receptor mRNA variants. Do they have a physiological role? Ann N Y Acad Sci. 1996 Apr 30;784:304–313. doi: 10.1111/j.1749-6632.1996.tb16245.x. [DOI] [PubMed] [Google Scholar]
  36. Pfeffer U., Fecarotta E., Arena G., Forlani A., Vidali G. Alternative splicing of the estrogen receptor primary transcript normally occurs in estrogen receptor positive tissues and cell lines. J Steroid Biochem Mol Biol. 1996 Jan;56(1-6):99–105. doi: 10.1016/0960-0760(95)00227-8. [DOI] [PubMed] [Google Scholar]
  37. Pichon M. F., Broet P., Magdelenat H., Delarue J. C., Spyratos F., Basuyau J. P., Saez S., Rallet A., Courriere P., Millon R. Prognostic value of steroid receptors after long-term follow-up of 2257 operable breast cancers. Br J Cancer. 1996 Jun;73(12):1545–1551. doi: 10.1038/bjc.1996.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Piva R., Bianchi N., Aguiari G. L., Gambari R., del Senno L. Sequencing of an RNA transcript of the human estrogen receptor gene: evidence for a new transcriptional event. J Steroid Biochem Mol Biol. 1993 Nov;46(5):531–538. doi: 10.1016/0960-0760(93)90179-z. [DOI] [PubMed] [Google Scholar]
  39. Sanger F., Coulson A. R. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol. 1975 May 25;94(3):441–448. doi: 10.1016/0022-2836(75)90213-2. [DOI] [PubMed] [Google Scholar]
  40. Thompson D. A., McPherson L. A., Carmeci C., deConinck E. C., Weigel R. J. Identification of two estrogen receptor transcripts with novel 5' exons isolated from a MCF7 cDNA library. J Steroid Biochem Mol Biol. 1997 Jun;62(2-3):143–153. doi: 10.1016/s0960-0760(97)00029-0. [DOI] [PubMed] [Google Scholar]
  41. Trask D. K., Band V., Zajchowski D. A., Yaswen P., Suh T., Sager R. Keratins as markers that distinguish normal and tumor-derived mammary epithelial cells. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2319–2323. doi: 10.1073/pnas.87.6.2319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Watson J. B., Margulies J. E. Differential cDNA screening strategies to identify novel stage-specific proteins in the developing mammalian brain. Dev Neurosci. 1993;15(2):77–86. doi: 10.1159/000111319. [DOI] [PubMed] [Google Scholar]
  43. Weigel R. J., deConinck E. C. Transcriptional control of estrogen receptor in estrogen receptor-negative breast carcinoma. Cancer Res. 1993 Aug 1;53(15):3472–3474. [PubMed] [Google Scholar]
  44. Williamson J. A., Bosher J. M., Skinner A., Sheer D., Williams T., Hurst H. C. Chromosomal mapping of the human and mouse homologues of two new members of the AP-2 family of transcription factors. Genomics. 1996 Jul 1;35(1):262–264. doi: 10.1006/geno.1996.0351. [DOI] [PubMed] [Google Scholar]
  45. Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994 Mar 3;368(6466):32–38. doi: 10.1038/368032a0. [DOI] [PubMed] [Google Scholar]
  46. Yaich L., Dupont W. D., Cavener D. R., Parl F. F. Analysis of the PvuII restriction fragment-length polymorphism and exon structure of the estrogen receptor gene in breast cancer and peripheral blood. Cancer Res. 1992 Jan 1;52(1):77–83. [PubMed] [Google Scholar]
  47. Zhao S., Ooi S. L., Pardee A. B. New primer strategy improves precision of differential display. Biotechniques. 1995 May;18(5):842-6, 848, 850. [PubMed] [Google Scholar]
  48. deConinck E. C., McPherson L. A., Weigel R. J. Transcriptional regulation of estrogen receptor in breast carcinomas. Mol Cell Biol. 1995 Apr;15(4):2191–2196. doi: 10.1128/mcb.15.4.2191. [DOI] [PMC free article] [PubMed] [Google Scholar]

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