Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Expression of Drosophila Ca2+ permeable transient receptor potential-like channel protein in a prostate cancer cell line decreases cell survival

Cancer Gene Therapy volume 10, pages 611–625 (2003)Cite this article

Abstract

The effects of expression of Drosophila melanoga ster Ca2+ permeable transient receptor potential-like (TRPL) channels, under the control of the cytomegalovirus (CMV) or prostate cell-specific promoters, on cell survival and apoptosis in the androgen-sensitive LNCaP prostate cancer cell line were investigated. A prostate-specific antigen (PSA) promoter construct (designated PSAEn/PSAPr) composed of a 0.6 kb region of the promoter and a 1.45 kb region of the enhancer resulted in androgen-dependent and prostate-specific expression of a luciferase reporter gene in transiently transfected LNCaP cells. Expression of the enhanced green fluorescence protein-TRPL chimeric protein under the control of the CMV promoter was confirmed by Western blot. Whereas the majority of the expressed protein was located in the cytoplasmic space, confocal microscopy with the CD-9 protein as a plasma membrane marker demonstrated that approximately 10% of the expressed TRPL protein was located in a band in the plasma membrane. Using recombinant adenoviruses, expression of the TRPL protein was associated with an increase in both the initial and sustained rates of Ca2+ inflow. Expression of TRPL under the control of the CMV promoter for 96 hours decreased cell number and increased the number of cells undergoing apoptosis by 23 and 27%, respectively. Apoptosis was inhibited by a caspase-3 inhibitor, Z-DEVD-fmk. It is concluded that, when heterologously expressed in LNCaP cells, the TRPL protein leads to a reduction in cell survival due, in part, to the induction of apoptosis. The effects of TRPL are likely caused by enhanced Na+ and Ca2+ inflow to the cells. This finding suggests a novel approach to modify the growth of prostate cancer cells that fail to undergo apoptosis following androgen ablation therapy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Scher HI, Steineck G, Kelly WK . Hormone-refractory (D3) prostate cancer: refining the concept. Urology. 1995;46:142–148.

    Article  CAS  PubMed  Google Scholar 

  2. Isaacs JT, Lundmo PI, Berges R, et al. Androgen regulation of programmed death of normal and malignant prostatic cells. J Androl. 1992;13:457–464.

    CAS  PubMed  Google Scholar 

  3. Gschwend JE . Apoptosis — principles and importance of programmed cell death for prostatic carcinoma. Urologe A. 1996;35:390–399.

    Article  CAS  PubMed  Google Scholar 

  4. Denmeade SR, Isaacs JT . Activation of programmed (apoptotic) cell death for the treatment of prostate cancer. Adv Pharmacol. 1996;35:281–306.

    Article  CAS  PubMed  Google Scholar 

  5. Gutierrez AA, Arias JM, Garcia L, et al. Activation of a Ca2+-permeable cation channel by two different inducers of apoptosis in a human prostatic cancer cell line. J Physiol. 1999;517:95–107.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Furuya Y, Lundmo P, Short AD, et al. The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin. Cancer Res. 1994;54: 6167–6175.

    CAS  PubMed  Google Scholar 

  7. Kyprianou NEHF, Isaacs JT . Activation of a Ca2+–Mg2+-dependent endonuclease as an early event in castration-induced prostatic cell death. Prostate. 1998;13:103–117.

    Article  Google Scholar 

  8. Thastrup O, Cullen PJ, Drobak BK, et al. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci USA. 1990;87:2466–2470.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Skryma R, Mariot P, Bourhis XL, et al. Store depletion and store-operated Ca2+ current in human prostate cancer LNCaP cells: involvement in apoptosis. J Physiol. 2000; 527(Part 1): 71–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Marcelli M, Cunningham GR, Haidacher SJ, et al. Caspase-7 is activated during lovastatin-induced apoptosis of the prostate cancer cell line LNCaP. Cancer Res. 1998;58:76–83.

    CAS  PubMed  Google Scholar 

  11. Lin XS, Denmeade SR, Cisek L, et al. Mechanism and role of growth arrest in programmed (apoptotic) death of prostatic cancer cells induced by thapsigargin. Prostate. 1997;33:201–207.

    Article  CAS  PubMed  Google Scholar 

  12. Martikainen P, Isaacs JT . Role of calcium in the programmed death of rat prostatic glandular cells. Prostate. 1990;17:175–187.

    Article  CAS  PubMed  Google Scholar 

  13. Phillips AM, Bull A, Kelly LE . Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene. Neuron. 1992;8:631–642.

    Article  CAS  PubMed  Google Scholar 

  14. Clapham DE, Runnels LW, Strubing C . The TRP ion channel family. Nat Rev Neurosci. 2001;2:387–396.

    Article  CAS  PubMed  Google Scholar 

  15. Lan L, Bawden MJ, Auld AM, et al. Expression of Drosophila trp1 cRNA in Xenopus laevis oocytes leads to the appearance of a Ca2+ channel activated by Ca2+ and calmodulin, and by guanosine 5′[gamma-thio]triphosphate. Biochem J. 1996;316:793–803.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gillo B, Chorna I, Cohen H, et al. Coexpression of Drosophila TRP and TRP-like proteins in Xenopus oocytes reconstitutes capacitative Ca2+ entry. Proc Natl Acad Sci USA. 1996;93:14146–14151.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zimmer S, Trost C, Wissenbach U, et al. Modulation of recombinant transient-receptor-potential-like (TRPL) channels by cytosolic Ca2+. Pflugers Arch. 2000;440:409–417.

    Article  CAS  PubMed  Google Scholar 

  18. Westfall MV, Rust EM, Albaya F, et al. Adenovirus-mediated myofilament gene transfer into adult cardiac myocytes. Meth Cell Biol. 1997;52:307–322.

    Article  CAS  Google Scholar 

  19. Thomsen DR, Stenberg RM, Goins WF, et al. Promoter-regulatory region of the major immediate early gene of human cytomegalovirus. Proc Natl Acad Sci USA. 1984;81:659–663.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. He TC, Zhou S, da Costa LT, et al. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA. 1998;95:2509–2514.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Niemeyer BA, Suzuki E, Scott K, et al. The Drosophila light-activated conductance is composed of the two channels TRP and TRPL. Cell. 1996;85:651–659.

    Article  CAS  PubMed  Google Scholar 

  22. Harlow E, Lane D . Antibodies: A Laboratory Manual. New York: Cold Spring Harbor Laboratories; 1988;313–315.

    Google Scholar 

  23. Xu HJ, Umapathysivam K, McNeilage J, et al. An enhanced chemiluminescence detection system combined with a modified immunoblot technique for the detection of low molecular weight IgM in sera from healthy adults and neonates. J Immunol Methods. 1992;146:241–247.

    Article  CAS  PubMed  Google Scholar 

  24. Imamura N, Mtasiwa DM, Ota H, et al. Distribution of cell surface glycoprotein CD9 (P24) antigen on megakaryocyte lineage leukemias and cell lines. Am J Hematol. 1990;35:65–67.

    Article  CAS  PubMed  Google Scholar 

  25. Grzegorz G, Poenie M, Tsien RY . A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440–3450.

    Google Scholar 

  26. Park JG, Kramer BS, Steinberg SM, et al. Chemosensitivity testing of human colorectal carcinoma cell lines using a tetrazolium-based colorimetric assay. Cancer Res. 1987;47:5875–5879.

    CAS  PubMed  Google Scholar 

  27. Ferguson DJ, Anderson TJ . Ultrastructural observations on cell death by apoptosis in the “resting” human breast. Virch Arch A Pathol Anat Histol. 1981;393:193–203.

    Article  CAS  Google Scholar 

  28. Yanagihara K, Tsumuraya M . Transforming growth factor beta 1 induces apoptotic cell death in cultured human gastric carcinoma cells. Cancer Res. 1992;52:4042–4045.

    CAS  PubMed  Google Scholar 

  29. Pang S, Taneja S, Dardashti K, et al. Prostate tissue specificity of the prostate-specific antigen promoter isolated from a patient with prostate cancer. Hum Gene Ther. 1995;6:1417–1426.

    Article  CAS  PubMed  Google Scholar 

  30. Schuur ER, Henderson GA, Kmetec LA, et al. Prostate-specific antigen expression is regulated by an upstream enhancer. J Biol Chem. 1996;271:7043–7051.

    Article  CAS  PubMed  Google Scholar 

  31. Rennie PS, Bruchovsky N, Leco KJ, et al. Characterization of two cis-acting DNA elements involved in the androgen regulation of the probasin gene. Mol Endocrinol. 1993;7:23–36.

    CAS  PubMed  Google Scholar 

  32. Tilley WD, Bentel JM, Aspinall JO, et al. Evidence for a novel mechanism of androgen resistance in the human prostate cancer cell line, PC-3. Steroids. 1995; 60:180–186.

    Article  CAS  PubMed  Google Scholar 

  33. Uchida S, Shimada Y, Watanabe G, et al. Motility-related protein (MRP-1/CD9) and KAI1/CD82 expression inversely correlate with lymph node metastasis in oesophageal squamous cell carcinoma. Br J Cancer. 1999;79:1168–1173.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chalfie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression. Science. 1994;263:802–805.

    Article  CAS  PubMed  Google Scholar 

  35. Strasser A, O'Connor L, Dixit VM . Apoptosis signaling. Ann Rev Biochem. 2000;69:217–245.

    Article  CAS  PubMed  Google Scholar 

  36. Coffey RN, Watson RW, Fitzpatrick JM . Signaling for the caspases: their role in prostate cell apoptosis. J Urol. 2001;165:5–14.

    Article  CAS  PubMed  Google Scholar 

  37. Cohen GM . Caspases: the executioners of apoptosis. Biochem J. 1997;326:1–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Alnemri ES, Livingston DJ, Nicholson DW, et al. Human ICE/CED-3 protease nomenclature. Cell. 1996;87:171.

    Article  CAS  PubMed  Google Scholar 

  39. Pastorino JG, Chen ST, Tafani M, et al. The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition. J Biol Chem. 1998;273:7770–7775.

    Article  CAS  PubMed  Google Scholar 

  40. Juin P, Pelletier M, Oliver L, et al. Induction of a caspase-3-like activity by calcium in normal cytosolic extracts triggers nuclear apoptosis in a cell-free system. J Biol Chem. 1998;273:17559–17564.

    Article  CAS  PubMed  Google Scholar 

  41. Reuss H, Mojet MH, Chyb S, et al. In vivo analysis of the Drosophila light-sensitive channels, TRP and TRPL. Neuron. 1997;19:1249–1259.

    Article  CAS  PubMed  Google Scholar 

  42. Gomez-Angelats M, Bortner CD, Cidlowski JA . Cell volume regulation in immune cell apoptosis. Cell Tissue Res. 2000;301:33–42.

    Article  CAS  PubMed  Google Scholar 

  43. Bortner CD, Cidlowski JA . Flow cytometric analysis of cell shrinkage and monovalent ions during apoptosis. Meths Cell Biol. 2001;66:49–67.

    Article  CAS  Google Scholar 

  44. Latham JP, Searle PF, Mautner V, et al. Prostate-specific antigen promoter/enhancer driven gene therapy for prostate cancer: construction and testing of a tissue-specific adenovirus vector. Cancer Res. 2000;60:334–341.

    CAS  PubMed  Google Scholar 

  45. Lee C, Sutkowski DM, Sensibar JA, et al. Regulation of proliferation and production of prostate-specific antigen in androgen-sensitive prostatic cancer cells, LNCaP, by dihydrotestosterone. Endocrinology. 1995;136:796–803.

    Article  CAS  PubMed  Google Scholar 

  46. Wang LG, Liu XM, Kreis W, et al. Phosphorylation/dephosphorylation of androgen receptor as a determinant of androgen agonistic or antagonistic activity. Biochem Biophys Res Commun. 1999;259:21–28.

    Article  CAS  PubMed  Google Scholar 

  47. Lan L, Brereton H, Barritt GJ . The role of calmodulin-binding sites in the regulation of the Drosophila TRPL cation channel expressed in Xenopus laevis oocytes by Ca2+, inositol 1,4,5-trisphosphate and GTP-binding proteins. Biochem J. 1998;330:1149–1158.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Bennett V, Lambert S, Davis JQ, et al. Molecular architecture of the specialized axonal membrane at the node of Ranvier. Soc Gen Physiol Ser. 1997;52:107–120.

    CAS  PubMed  Google Scholar 

  49. Bourguignon LY, Chu A, Jin H, et al. Ryanodine receptor-ankyrin interaction regulates internal Ca2+ release in mouse T-lymphoma cells. J Biol Chem. 1995;270:17917–17922.

    Article  CAS  PubMed  Google Scholar 

  50. Bourguignon LY, Jin H, Ida N, et al. The involvement of ankyrin in regulation of inositol 1,4,5 trisphosphate receptor-mediated internal Ca2+ release from Ca2+ storage vehicles ion mouse T-lymphoma cells. J Biol Chem. 1993;268:7280–7297.

    Google Scholar 

  51. Drenckhahn D, Bennett V . Polarized distribution of Mr 210,000 and 190,000 analogs of erythrocyte ankyrin along the plasma membrane of transporting epithelia, neurons and photoreceptors. Eur J Cell Biol. 1987;43:479–486.

    CAS  PubMed  Google Scholar 

  52. Bahri SM, Yang X, Chia W . The Drosophila biofocal gene encodes a novel protein which co-localizes with actin and is necessary for photoreceptor morphogenesis. Mol Cell Biol. 1997;17:5521–5529.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Welsh CF, Zhu D, Bourguigon LY . Interaction of CD44 variant isoforms with hyaluronic acid and the cytoskeleton in human prostate cancer cells. J Cell Physiol. 1995;164:605–612.

    Article  CAS  PubMed  Google Scholar 

  54. Horoszewicz JS, Leong SS, Chu TM, et al. The LNCaP cell line — a new model for studies on human prostatic carcinoma. Prog Clin Biol Res. 1980;37:115–132.

    CAS  PubMed  Google Scholar 

  55. Wang W, O'Connell B, Dykeman R, et al. Cloning of TRP1 beta isoform from rat brain: immunodetection and localisation of the endogenous TRP1 protein. Am J Physiol 1999;276:C969–C979.

    Article  CAS  PubMed  Google Scholar 

  56. Bahner M, Frechter S, Silva ND, et al. Light-regulated subcellular translocation of Drosophila TRPL channels induces long-term adaptation and modifies the light-induced current. Neuron. 2002;34:83–93.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors are grateful to Mrs Diana Kassos for typing the manuscript, Ms Jenny Hiscock for assistance with the confocal microscopy, Mr Miao Yang for advice on prostate cell-specific promoters, the Anti-Cancer Foundation of the Universities of South Australia for grant support, and to the Australian Government for the provision of an International Postgraduate Research Scholarship to Lei Zhang.

Author information

Authors and Affiliations

  1. Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, GPO Box 2100, Adelaide, 5001, South Australia, Australia

    Lei Zhang, Helen M Brereton, Michael Hahn, Mario Froscio & Greg J Barritt

  2. Department of Surgery, School of Medicine, Faculty of Health Sciences, Flinders University, GPO Box 2100, Adelaide, 5001, South Australia, Australia

    Wayne D Tilley

  3. Medical Oncology, Level 7 East Wing, RAH Cancer Centre, Royal Adelaide Hospital, North Terrace, Adelaide, 5000, South Australia, Australia

    Michael P Brown

Authors
  1. Lei Zhang
    View author publications

    Search author on:PubMed Google Scholar

  2. Helen M Brereton
    View author publications

    Search author on:PubMed Google Scholar

  3. Michael Hahn
    View author publications

    Search author on:PubMed Google Scholar

  4. Mario Froscio
    View author publications

    Search author on:PubMed Google Scholar

  5. Wayne D Tilley
    View author publications

    Search author on:PubMed Google Scholar

  6. Michael P Brown
    View author publications

    Search author on:PubMed Google Scholar

  7. Greg J Barritt
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Greg J Barritt.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, L., Brereton, H., Hahn, M. et al. Expression of Drosophila Ca2+ permeable transient receptor potential-like channel protein in a prostate cancer cell line decreases cell survival. Cancer Gene Ther 10, 611–625 (2003). https://doi.org/10.1038/sj.cgt.7700608

Download citation

  • Received:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/sj.cgt.7700608

Keywords

This article is cited by

Search

Advanced search

Quick links