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Molecular mechanisms regulating the tumor-targeting potential of splice-activated gene expression

Cancer Gene Therapy volume 11, pages 797–807 (2004)Cite this article

Abstract

Previous studies have suggested that differences in the ability of normal and malignant cells to process certain alternatively spliced pre-mRNA transcripts can be exploited as a potentially powerful means of targeting the expression of therapeutic genes to tumor cells in vivo and in vitro. Specifically, it was shown that efficient processing of minigene constructs containing the alternatively spliced CD44 exons v9 and v10 only occurs in tumor cells that express CD44 isoforms that incorporate these exons (e.g. CD44R1). In the present study, efforts were made to define the molecular mechanisms that underlie the apparent specificity of this process. RT-PCR analysis and DNA sequencing were used to characterize the various splicing events that occur between CD44 exons v8, v9 and v10 following transfection of minigene constructs containing these various exons into CD44R1-positive (PC3) and CD44R1-negative (T24) cell lines. The results obtained confirm that although the v8–v9 intron is efficiently removed in both CD44R1-positive and CD44R1-negative cells, the corresponding v9–v10 intron is accurately spliced and the exons appropriately joined only in lines that express v10-containing CD44 isoforms (e.g. PC3). In CD44R1-negative cell lines (e.g. T24) alternative 5′ and 3′ splice sites located within the v9–v10 intron are preferentially used, resulting in various portions of the intron being retained within the final processed mRNA product. It is proposed that identification of these functionally important intronic sequence elements will facilitate the development of second generation “splice activated gene expression” vectors that may prove useful in various cancer gene therapy applications.

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References

  1. Green NK, Seymour LW . Adenoviral vectors: systemic delivery and tumor targeting. Cancer Gene Ther. 2002;9:1036–1042.

    Article  CAS  Google Scholar 

  2. Greco O, Marples B, Dachs GU, et al. Novel chimeric gene promoters responsive to hypoxia and ionizing radiation. Gene Therapy. 2002;9:1403–1411.

    Article  CAS  Google Scholar 

  3. Greco O, Scott SD, Marples B, et al. Cancer gene therapy: ‘delivery, delivery, delivery’. Front Biosci. 2002;7:d1516–d1524.

    PubMed  Google Scholar 

  4. Hayes GM, Carpenito C, Davis PD, et al. Alternative splicing as a novel of means of regulating the expression of therapeutic genes. Cancer Gene Ther. 2002;9:133–141.

    Article  CAS  Google Scholar 

  5. Dreyfuss G, Hentze M, Lamond AI . From transcript to protein. Cell. 1996;85:963–972.

    Article  CAS  Google Scholar 

  6. Lopez AJ . Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation. Annu Rev Genet. 1998;32:279–305.

    Article  CAS  Google Scholar 

  7. Cooper DL, Dougherty GJ . To metastasize or not? Selection of CD44 splice sites. Nat Med. 1995;1:635–637.

    Article  CAS  Google Scholar 

  8. Naot D, Sionov RV, Ish-Shalom D . CD44: structure, function, and association with the malignant process. Adv Cancer Res. 1997;71:241–319.

    Article  Google Scholar 

  9. Dougherty GJ, Landorp PM, Cooper DL, et al. Molecular cloning of CD44R1 and CD44R2, two novel isoforms of the human CD44 lymphocyte “homing” receptor expressed by hemopoietic cells. J Exp Med. 1991;174:1–5.

    Article  CAS  Google Scholar 

  10. Sneath RJ, Mangham DC . The normal structure and function of CD44 and its role in neoplasia. Mol Pathol. 1998;51:191–200.

    Article  CAS  Google Scholar 

  11. Chiu RK, Droll A, Dougherty ST, et al. Alternatively spliced CD44 isoforms containing exon v10 promote cellular adhesion through the recognition of chondroitin sulfate-modified CD44. Exp Cell Res. 1999;248:314–321.

    Article  CAS  Google Scholar 

  12. Kaighn ME, Lechner JF, Narayan KS, et al. Prostate carcinoma: tissue culture cell lines. Natl Cancer Inst Monogr. 1978;49:17–21.

    Google Scholar 

  13. Kaighn ME, Narayan KS, Ohnuki Y, et al. Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest Urol. 1979;17:16–23.

    CAS  PubMed  Google Scholar 

  14. O'Toole C, Perlmann P, Unsgaard B, et al. Cellular immunity to human urinary bladder carcinoma. I. Correlation to clinical stage and radiotherapy. Int J Cancer. 1972;10:77–91.

    Article  CAS  Google Scholar 

  15. Dougherty GJ, Cooper DL, Memory JF, et al. Ligand binding specificity of alternatively spliced CD44 isoforms. Recognition and binding of hyaluronan by CD44R1. J Biol Chem. 1994;269:9074–9078.

    CAS  PubMed  Google Scholar 

  16. Fu XD . The superfamily of arginine/serine-rich splicing factors. RNA. 1995;1:663–680.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Adams MD, Rudner DZ, Rio DC . Biochemistry and regulation of pre-mRNA splicing. Curr Opin Cell Biol. 1996;8:331–339.

    Article  CAS  Google Scholar 

  18. Mayeda A, Krainer AR . Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. Cell. 1992;68:365–375.

    Article  CAS  Google Scholar 

  19. Mayeda A, Munroe SH, Caceres JF, et al. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J. 1994;13:5483–5495.

    Article  CAS  Google Scholar 

  20. Smith CW, Valcarcel J . Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem Sci. 2000;25:381–388.

    Article  CAS  Google Scholar 

  21. Screaton GR, Caceres JF, Mayeda A, et al. Identification and characterization of three members of the human SR family of pre-mRNA splicing factors. EMBO J. 1995;14:4336–4349.

    Article  CAS  Google Scholar 

  22. Galiana-Arnoux D, Lejeune F, Gesnel MC, et al. The CD44 alternative v9 exon contains a splicing enhancer responsive to the SR proteins 9G8, ASF/SF2, and SRp20. J Biol Chem. 2003;278:32943–32953.

    Article  CAS  Google Scholar 

  23. Konig H, Ponta H, Herrlich P . Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator. EMBO J. 1998;17:2904–2913.

    Article  CAS  Google Scholar 

  24. Bell MV, Cowper AE, Lefranc MP, et al. Influence of intron length on alternative splicing of CD44. Mol Cell Biol. 1998;18:5930–5941.

    Article  CAS  Google Scholar 

  25. Yu Q, Toole BP . A new alternatively spliced exon between v9 and v10 provides a molecular basis for synthesis of soluble CD44. J Biol Chem. 1996;271:20603–20607.

    Article  CAS  Google Scholar 

  26. Mironov AA, Fickett JW, Gelfand MS . Frequent alternative splicing of human genes. Genome Res. 1999;9:1288–1293.

    Article  CAS  Google Scholar 

  27. Hanke J, Brett D, Zastrow I, et al. Alternative splicing of human genes: more the rule than the exception? Trends Genet. 1999;15:389–390.

    Article  CAS  Google Scholar 

  28. Caballero OL, de SSJ, Brentani RR, et al. Alternative spliced transcripts as cancer markers. Dis Markers. 2001;17:67–75.

    Article  CAS  Google Scholar 

  29. Chiu RK, Carpenito C, Dougherty ST, et al. Identification and characterization of CD44RC, a novel alternatively spliced soluble CD44 isoform that can potentiate the hyaluronan binding activity of cell surface CD44. Neoplasia. 1999;1:446–452.

    Article  CAS  Google Scholar 

  30. Tasch J, Gong M, Sadelain M, et al. A unique folate hydrolase, prostate-specific membrane antigen (PSMA): a target for immunotherapy? Crit Rev Immunol. 2001;21:249–261.

    Article  CAS  Google Scholar 

  31. Kong QY, Liu J, Chen XY, et al. Differential expression patterns of hyaluronan receptors CD44 and RHAMM in transitional cell carcinomas of urinary bladder. Oncol Rep. 2003;10:51–55.

    CAS  PubMed  Google Scholar 

  32. Toran-Allerand CD . Mini-review: a plethora of estrogen receptors in the brain: where will it end? Endocrinology. 2004;145:1069–1074.

    Article  CAS  Google Scholar 

  33. Leung DW, Tompkins CK, White T . Characterization of two spliced variants of human phosphatidic acid phosphatase cDNAs that are differentially expressed in normal and tumor cells. Adv Exp Med Biol. 1999;469:639–646.

    Article  CAS  Google Scholar 

  34. Nichols SE, Harper DC, Berson JF, et al. A novel splice variant of Pmel17 expressed by human melanocytes and melanoma cells lacking some of the internal repeats. J Invest Dermatol. 2003;121:821–830.

    Article  CAS  Google Scholar 

  35. Lee JH, Seo YW, Park SR, et al. Expression of a splice variant of KAI1, a tumor metastasis suppressor gene, influences tumor invasion and progression. Cancer Res. 2003;63:7247–7255.

    CAS  PubMed  Google Scholar 

  36. Lu F, Gladden AB, Diehl JA . An alternatively spliced cyclin D1 isoform, cyclin D1b, is a nuclear oncogene. Cancer Res. 2003;63:7056–7061.

    CAS  PubMed  Google Scholar 

  37. Philips AV, Cooper TA . RNA processing and human disease. Cell Mol Life Sci. 2000;57:235–249.

    Article  CAS  Google Scholar 

  38. Sampath J, Long PR, Shepard RL, et al. Human SPF45, a splicing factor, has limited expression in normal tissues, is overexpressed in many tumors, and can confer a multidrug-resistant phenotype to cells. Am J Pathol. 2003;163:1781–1790.

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported, in part, by Grant RO1 CA100004 from the National Institutes of Health.

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Authors and Affiliations

  1. Department of Radiation Oncology, David Geffen School of Medicine at UCLA, Los Angeles, California, USA

    Gregory M Hayes & Graeme J Dougherty

  2. Department of Radiation Oncology, University of Arizona, Tucson, Arizona, USA

    Shona T Dougherty

  3. Angiogene Pharmaceuticals Ltd, The Magdalen Centre, Oxford Science Park, Oxford, UK

    Peter D Davis

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  1. Gregory M Hayes
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  2. Shona T Dougherty
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  3. Peter D Davis
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  4. Graeme J Dougherty
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Correspondence to Graeme J Dougherty.

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Hayes, G., Dougherty, S., Davis, P. et al. Molecular mechanisms regulating the tumor-targeting potential of splice-activated gene expression. Cancer Gene Ther 11, 797–807 (2004). https://doi.org/10.1038/sj.cgt.7700759

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