Search tips
Search criteria 


Logo of molmedLink to Publisher's site
Mol Med. 1999 August; 5(8): 526–541.
PMCID: PMC2230455

Re-expression of SPR1 in breast cancer cells by phorbol 12-myristate 13-acetate (PMA) or UV irradiation is mediated by the AP-1 binding site in the SPR1 promoter.


BACKGROUND: Invasive tumor cells are characterized by multiple phenotypic changes as a result of the large number of cDNAs being differentially expressed in tumor cells compared to normal progenitors. Expression genetics focuses on changes at the RNA level with the aim of identifying functionally important genes whose aberrant expression in cancer cells is regulated at the level of transcription. These genes were named class II genes and are distinguished from class I genes, which are characterized by genomic mutations, deletions, or other alterations. Reversal of the tumor cell phenotype accompanying normalization of the expression of such genes may be exploited therapeutically if gene expression can be specifically modulated by drugs or other treatments. Considering that genes are coordinately regulated in complex networks, it is likely that the expression of multiple genes can be simultaneously modulated in tumor cells by drugs acting on the signal transduction pathway that regulates their expression. The SPR1 gene is associated with differentiation and its expression is down-regulated or inactivated in malignant cells. Analysis of the SPR1 promoter showed that down-regulation of SPR1 expression in breast tumor cells occurs at the level of transcription. SPR1 presents an example of class II genes, since its expression was up-regulated in tumor cells by phorbol 12-myristate 13-acetate (PMA) or by ultraviolet (UV) irradiation. MATERIALS AND METHODS: The SPR1 gene was identified by differential display on the basis of its reduced or absent expression in human breast tumor cell lines compared to normal mammary epithelial cell strains. Differential expression was confirmed by Northern blot analysis employing multiple normal and tumor cell lines. The promoter region -619 to +15 of the SPR1 gene was sequenced and analyzed by CAT assays, deletion analysis, and mutagenesis. Up-regulation of SPR1 expression by PMA and UV irradiation was monitored by Northern analysis and analyzed by CAT assays. RESULTS: The mechanism of down-regulation of SPR1 expression in breast tumor cells was investigated. It was found that the -619 to +15 upstream promoter region is sufficient for SPR1 expression in normal breast cells, but it is transcriptionally silent in most breast tumor cell lines. By deletion analysis and mutagenesis, two upstream cis-acting promoter elements were identified. Our data indicate that the AP-1 element located between -139 and -133 acts as a major enhancer of SPR1 transcription only in normal mammary epithelial cells but not in corresponding tumor cells, whereas the sequences flanking the AP-1 site do not affect its promoter enhancing activity. In addition, a transcriptional repressor was identified that binds unknown factor(s) and is active in both normal and tumor breast cells. Inhibitor function was mapped to a 35-bp element located from -178 to -139 upstream of the human SPR1 mRNA start site. The expression of SPR1 could be induced in the 21MT-2 metastatic breast tumor cell line by PMA treatment or by short UV irradiation via a transcriptional mechanism. AP-1 is the cis element mediating the transcriptional activation of SPR1 by PMA, which induces the expression of AP-1 factors in 21MT-2 cells. Mutation of the AP-1 site abolishes the induction of SPR1 expression by PMA. CONCLUSIONS: Our results demonstrate that loss of SPR1 expression in breast tumor cells results from impaired transactivation through the AP-1 site in the SPR1 promoter, as well as from the presence of a negative regulatory element active in both normal and tumor cells. Furthermore, our results provide a basis for therapeutic manipulation of down-regulated genes, such as SPR1, in human cancers.

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 (3.0M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Sager R. Expression genetics in cancer: shifting the focus from DNA to RNA. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):952–955. [PubMed]
  • Zou Z, Anisowicz A, Hendrix MJ, Thor A, Neveu M, Sheng S, Rafidi K, Seftor E, Sager R. Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science. 1994 Jan 28;263(5146):526–529. [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]
  • 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]
  • Sager R, Sheng S, Anisowicz A, Sotiropoulou G, Zou Z, Stenman G, Swisshelm K, Chen Z, Hendrix MJ, Pemberton P, et al. RNA genetics of breast cancer: maspin as paradigm. Cold Spring Harb Symp Quant Biol. 1994;59:537–546. [PubMed]
  • Sotiropoulou G, Anisowicz A, Sager R. Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer. J Biol Chem. 1997 Jan 10;272(2):903–910. [PubMed]
  • Anisowicz A, Sotiropoulou G, Stenman G, Mok SC, Sager R. A novel protease homolog differentially expressed in breast and ovarian cancer. Mol Med. 1996 Sep;2(5):624–636. [PMC free article] [PubMed]
  • Zhang L, Zhou W, Velculescu VE, Kern SE, Hruban RH, Hamilton SR, Vogelstein B, Kinzler KW. Gene expression profiles in normal and cancer cells. Science. 1997 May 23;276(5316):1268–1272. [PubMed]
  • Kartasova T, van de Putte P. Isolation, characterization, and UV-stimulated expression of two families of genes encoding polypeptides of related structure in human epidermal keratinocytes. Mol Cell Biol. 1988 May;8(5):2195–2203. [PMC free article] [PubMed]
  • Kartasova T, van Muijen GN, van Pelt-Heerschap H, van de Putte P. Novel protein in human epidermal keratinocytes: regulation of expression during differentiation. Mol Cell Biol. 1988 May;8(5):2204–2210. [PMC free article] [PubMed]
  • Gibbs S, Fijneman R, Wiegant J, van Kessel AG, van De Putte P, Backendorf C. Molecular characterization and evolution of the SPRR family of keratinocyte differentiation markers encoding small proline-rich proteins. Genomics. 1993 Jun;16(3):630–637. [PubMed]
  • Tesfaigzi J, An G, Wu R, Carlson DM. Two nuclear proteins in tracheal epithelial cells are recognized by antibodies specific to a squamous differentiation marker, sprI. J Cell Physiol. 1995 Sep;164(3):571–578. [PubMed]
  • Tesfaigzi J, Carlson DM. Expression of the spr1 gene in cultured tracheal epithelial cells and its regulation by retinoids before and after confluence. J Cell Physiol. 1996 Mar;166(3):480–486. [PubMed]
  • An G, Tesfaigzi J, Chuu YJ, Wu R. Isolation and characterization of the human spr1 gene and its regulation of expression by phorbol ester and cyclic AMP. J Biol Chem. 1993 May 25;268(15):10977–10982. [PubMed]
  • Reddy SP, Chuu YJ, Lao PN, Donn J, Ann DK, Wu R. Expression of human squamous cell differentiation marker, SPR1, in tracheobronchial epithelium depends on JUN and TRE motifs. J Biol Chem. 1995 Nov 3;270(44):26451–26459. [PubMed]
  • Band V, Zajchowski D, Swisshelm K, Trask D, Kulesa V, Cohen C, Connolly J, Sager R. Tumor progression in four mammary epithelial cell lines derived from the same patient. Cancer Res. 1990 Nov 15;50(22):7351–7357. [PubMed]
  • Band V, Sager R. Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1249–1253. [PubMed]
  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Sassone-Corsi P, Verma IM. Modulation of c-fos gene transcription by negative and positive cellular factors. Nature. 1987 Apr 2;326(6112):507–510. [PubMed]
  • Anisowicz A, Messineo M, Lee SW, Sager R. An NF-kappa B-like transcription factor mediates IL-1/TNF-alpha induction of gro in human fibroblasts. J Immunol. 1991 Jul 15;147(2):520–527. [PubMed]
  • Graham FL, van der Eb AJ. Transformation of rat cells by DNA of human adenovirus 5. Virology. 1973 Aug;54(2):536–539. [PubMed]
  • Fridovich-Keil JL, Gudas JM, Dou QP, Bouvard I, Pardee AB. Growth-responsive expression from the murine thymidine kinase promoter: genetic analysis of DNA sequences. Cell Growth Differ. 1991 Feb;2(2):67–76. [PubMed]
  • Gorman CM, Moffat LF, Howard BH. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. [PMC free article] [PubMed]
  • Chan GL, Little JB. Induction of oncogenic transformation in vitro by ultraviolet light. Nature. 1976 Dec 2;264(5585):442–444. [PubMed]
  • Luckow B, Schütz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 1987 Jul 10;15(13):5490–5490. [PMC free article] [PubMed]
  • Zhang M, Maass N, Magit D, Sager R. Transactivation through Ets and Ap1 transcription sites determines the expression of the tumor-suppressing gene maspin. Cell Growth Differ. 1997 Feb;8(2):179–186. [PubMed]
  • Sutherland JA, Cook A, Bannister AJ, Kouzarides T. Conserved motifs in Fos and Jun define a new class of activation domain. Genes Dev. 1992 Sep;6(9):1810–1819. [PubMed]
  • Halazonetis TD, Georgopoulos K, Greenberg ME, Leder P. c-Jun dimerizes with itself and with c-Fos, forming complexes of different DNA binding affinities. Cell. 1988 Dec 2;55(5):917–924. [PubMed]
  • Zhang M, Magit D, Pardee AB, Sager R. Re-expression of elafin in 21MT2 breast carcinomas by phorbol 12-myristate 13-acetate is mediated by the Ap1 site in the elafin promoter. Cancer Res. 1997 Oct 15;57(20):4631–4636. [PubMed]
  • Binétruy B, Smeal T, Karin M. Ha-Ras augments c-Jun activity and stimulates phosphorylation of its activation domain. Nature. 1991 May 9;351(6322):122–127. [PubMed]
  • Dong Z, Birrer MJ, Watts RG, Matrisian LM, Colburn NH. Blocking of tumor promoter-induced AP-1 activity inhibits induced transformation in JB6 mouse epidermal cells. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):609–613. [PubMed]
  • Li JJ, Westergaard C, Ghosh P, Colburn NH. Inhibitors of both nuclear factor-kappaB and activator protein-1 activation block the neoplastic transformation response. Cancer Res. 1997 Aug 15;57(16):3569–3576. [PubMed]
  • Domann FE, Jr, Levy JP, Finch JS, Bowden GT. Constitutive AP-1 DNA binding and transactivating ability of malignant but not benign mouse epidermal cells. Mol Carcinog. 1994 Feb;9(2):61–66. [PubMed]
  • Tyrrell RM. Activation of mammalian gene expression by the UV component of sunlight--from models to reality. Bioessays. 1996 Feb;18(2):139–148. [PubMed]
  • Gibbs S, Lohman F, Teubel W, van de Putte P, Backendorf C. Characterization of the human spr2 promoter: induction after UV irradiation or TPA treatment and regulation during differentiation of cultured primary keratinocytes. Nucleic Acids Res. 1990 Aug 11;18(15):4401–4407. [PMC free article] [PubMed]
  • Büscher M, Rahmsdorf HJ, Litfin M, Karin M, Herrlich P. Activation of the c-fos gene by UV and phorbol ester: different signal transduction pathways converge to the same enhancer element. Oncogene. 1988 Sep;3(3):301–311. [PubMed]
  • Zhang M, Martin KJ, Sheng S, Sager R. Expression genetics: a different approach to cancer diagnosis and prognosis. Trends Biotechnol. 1998 Feb;16(2):66–71. [PubMed]

Articles from Molecular Medicine are provided here courtesy of The Feinstein Institute for Medical Research at North Shore LIJ