Journal of Neuro-Oncology

, Volume 118, Issue 1, pp 29–37 | Cite as

Increased sensitivity of glioma cells to 5-fluorocytosine following photo-chemical internalization enhanced nonviral transfection of the cytosine deaminase suicide gene

  • Frederick WangEmail author
  • Genesis Zamora
  • Chung-Ho Sun
  • Anthony Trinidad
  • Changho Chun
  • Young Jik Kwon
  • Kristian Berg
  • Steen J. Madsen
  • Henry Hirschberg
Laboratory Investigation


Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80 % of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments.PCI was performed with the photosensitizer AlPcS2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect.


Glioma Gene Therapy PDT PCI Cytosine deaminase 5-FC 



This work was supported by grants from the Norwegian Radium Hospital Research Foundation and the Chao Cancer Center. Portions of this work were made possible through access to the Laser Microbeam and Medical Program (LAMMP) and the Chao Cancer Center Optical Biology Shared Resource at the University of California, Irvine.


  1. 1.
    Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups, National Cancer Institute of Canada Clinical Trials Group et al (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996PubMedCrossRefGoogle Scholar
  2. 2.
    Brandes AA, Tosoni A, Franceschi E, Reni M, Gatta G, Vecht C (2008) Glioblastoma in adults. Crit Rev Oncol Hematol 67:139–152PubMedCrossRefGoogle Scholar
  3. 3.
    Petrecca K, Guiot MC, Panet-Raymond V, Souhami L (2013) Failure pattern following complete resection plus radiotherapy and temozolomide is at the resection margin in patients with Glioblastoma. J Neurooncol 111:19–23PubMedCrossRefGoogle Scholar
  4. 4.
    Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM (1992) In vivo gene transfer with retroviral-vector-producer cells for treatment of experimental brain tumors. Science 256:1550–1552PubMedCrossRefGoogle Scholar
  5. 5.
    Moolton F (1986) Tumor chemosensitivity conferred by inserted herpes thymidine-kinase genes: paradigm for a prospective cancer-control strategy. Cancer Res 46:5276–5281Google Scholar
  6. 6.
    Mullen CA, Coale MM, Lowe R, Blase RM (1994) Tumors expressing the cytosine-deaminase suicide gene can be eliminated in vivo with 5-fluorocytosine and induce protective immunity to wild-type tumor. Cancer Res 54:1503–1506PubMedGoogle Scholar
  7. 7.
    Kai G, Lingfei X, Zhongcheng Z, Dehua X, Lanyin S, Xinyuan L (1997) Transduction of cytosine deaminase gene makes rat gliomas cells highly sensitive to 5-fluorocytosine. Int J Cancer 71:675–679CrossRefGoogle Scholar
  8. 8.
    Miller RC, Williams CR, Buchsbaum DJ, Gillespie GY (2002) Intratumoral 5-fluorouracil produced by cytosine deaminase/5-fluorocytosine gene therapy is effective for experimental human glioblastomas. Cancer Res 62:773–780PubMedGoogle Scholar
  9. 9.
    Ostertag D, Amundson KK, Espinoza FL, Martin B, Buckley T, da Silva G et al (2012) Brain tumor eradication and prolonged survival from intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a non lytic retroviral replicating vector. Neuro-oncology 14:145–159PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Aboody KS, Najbauer J, Metz MZ, D’Apuzzo M, Gutova M, Annala AJ, Synold TW, Couture LA, Blanchard S, Moats RA, Garcia E, Aramburo S, Valenzuela VV, Frank RT, Barish ME, Brown CE, Kim SU, Badie B, Portnow J (2013) Neural stem cell-mediated enzyme-prodrug therapy for glioma: preclinical studies. Transl Med Sci. doi: 10.1126/scitranslmed.3005365 Google Scholar
  11. 11.
    Perez OD, Logg CR, Hiraoka K et al (2012) Design and selection of Toca 511 for clinical use: modified retroviral replicating vector with improved stability and gene expression. Mol Ther. doi: 10.1038/mt.2012.83 Google Scholar
  12. 12.
    Paar M, Schwab S, Rosenfellner D, Salmons B, Gunzburg WH, Renner M et al (2007) Effects of viral strain, transgene position, and target cell type on replication kinetics, genomic stability, and transgene expression of replication-competent murine leukemia virus-based vectors. J Virol 81:6973–6983PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Liu J, Guo S, Li Z, Liu L, Gu J (2009) Synthesis and characterization of stearyl protamine and investigation of their complexes with DNA for gene delivery. Colloids Surf B Biointerfaces. doi: 10.1016/j.colsurfb.2009.04.026 Google Scholar
  14. 14.
    Sun X, Zhang N (2010) Cationic Polymer Optimization for Efficient Gene Delivery. Mini Rev Med Chem 10:108–125PubMedCrossRefGoogle Scholar
  15. 15.
    Høgset A, Engesaeter BO, Prasmickaite L, Berg K, Fodstad O, Maelandsmo GM (2002) Light induced adenovirus gene transfer, an efficient and specific gene delivery technology for cancer gene therapy. Cancer Gene Ther 9:365–371PubMedCrossRefGoogle Scholar
  16. 16.
    Berg K, Berstad M, Prasmickaite L, Weyergang A, Selbo PK, Hedfors I, Høgset A (2010) Photochemical internalization (PCI): a new tool for gene and oligonucleotide delivery. Top Curr Chem 296:251–281PubMedCrossRefGoogle Scholar
  17. 17.
    Selbo PK, Weyergang A, Høgset A, Norum OJ, Berstad MB, Vikdal M, Berg K (2010) Photochemical internalization provides time and space-controlled endolysosomal escape of therapeutic molecules. J. Control Release 148:2–12. doi: 10.1016/j.jconrel.2010.06.008 PubMedCrossRefGoogle Scholar
  18. 18.
    Maurice-Duelli A, Ndoye A, Bouali S, Leroux A, Merlin JL (2004) Enhanced cell growth inhibition following PTEN nonviral gene transfer using polyethylenimine and photochemical internalization in endometrial cancer cells. Technol Cancer Res Treat 3:459–465PubMedGoogle Scholar
  19. 19.
    Ndoye A, Dolivet G, Høgset A, Leroux A, Fifre A, Erbacher P, Berg K, Behr JP, Guillemin F, Merlin JL (2006) Eradication of p53-mutated head and neck squamous cell carcinoma xenografts using nonviral p53 gene therapy and photochemical internalization. Mol Ther 13:1156–1162PubMedCrossRefGoogle Scholar
  20. 20.
    Mathews MS, Shih EC, Zamora G, Sun CH, Cho SK, Kwon YJ, Hirschberg H (2012) Glioma cell growth inhibition following photochemical internalization enhanced non-viral PTEN gene transfection. Lasers Surg Med 44:746–754. doi: 10.1002/lsm.22082 PubMedCrossRefGoogle Scholar
  21. 21.
    Prasmickaite L, Høgset A, Olsen VM, Kaalhus O, Mikalsen SO, Berg K (2004) Photochemically enhanced gene transfection increases the cytotoxicity of the herpes simplex virus thymidine kinase gene combined with ganciclovir. Cancer Gene Ther 11:514–523PubMedCrossRefGoogle Scholar
  22. 22.
    Madsen SJ, Sun CH, Tromberg BJ, Cristini V, De Magalhães N, Hirschberg H (2006) Multicell tumor spheroids in photodynamic therapy. Lasers Surg Med 38:555–564PubMedCrossRefGoogle Scholar
  23. 23.
    Ivascu A, Kubbies M (2006) Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J Biomol Screen 11:922–932PubMedCrossRefGoogle Scholar
  24. 24.
    Mathews MS, Blickenstaff JW, Shih EC, Zamora G, Vo V, Sun CH, Hirschberg H, Madsen SJ (2012) Photochemical internalization of bleomycin for glioma treatment. J Biomed Opt 17:058001. doi: 10.1117/1.JBO.17.5.058001 PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Adachi Y, Tamiya T, Ichikawa T, Terada K, Ono Y, Matsumoto K, Furuta T, Hamada H, Ohmoto T (2000) Experimental gene therapy for brain tumors using adenovirus-mediated transfer of cytosine deaminase gene and uracil phosphoribosyltransferase gene with 5-fluorocytosine. Hum Gene Ther 11:77–89PubMedCrossRefGoogle Scholar
  26. 26.
    Erbs P, Regulier E, Kintz J, Leroy P, Poitevin Y, Exinger F, Jund R, Mehtali M (2000) In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene. Cancer Res 60:3813–3822PubMedGoogle Scholar
  27. 27.
    Rainov NG (2000) A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme. Hum Gene Ther 11:2389–2401PubMedCrossRefGoogle Scholar
  28. 28.
    FassRJ Perkins RL (1971) 5-fluorocytosine in the treatment of cryptococcal and candida mycoses. Ann Intern Med 74:535–539. doi: 10.7326/0003-4819-74-4-535 CrossRefGoogle Scholar
  29. 29.
    Cutler RE, Blair AD, Kelly MR (1978) Flucytosine kinetics in subjects with normal and impaired renal function. Clin Pharmacol Ther 24:333–342PubMedGoogle Scholar
  30. 30.
    Morse GD, Shelton MJ, O’Donnell AM (1993) Comparative pharmacokinetics of antiviral nucleoside analogues. Clin Pharmacokinet 24:101–123PubMedCrossRefGoogle Scholar
  31. 31.
    Brewester ME, Raghavan K, Pop E, Bodor N (1994) Enhanced delivery of ganciclovir to the brain through the use of redox targeting. Antimicrob Agents Chemother 38:817–823CrossRefGoogle Scholar
  32. 32.
    Barriere SL (1990) Pharmacology and pharmacokinetics of traditional systemicantifungal agents. Pharmacotherapy 10:134S–140S. doi: 10.1002/j.1875-9114.1990.tb02598.x PubMedGoogle Scholar
  33. 33.
    Huber BE, Austin EA, Richards CA, Davis ST, Good SS (1994) Metabolism of 5-fluorocytosine to 5 fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc Natl Acad Sci USA 91:8302–8306PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Williams CW, Buchsbaum DJ, Miller CR (2001) Uracil phosphoribosyltransferase potentiates 5-fluorouracil and cytosine deaminase/5-fluorocytosine cytotoxicity in prostate cancer. Proc Am Assoc Cancer Res 42:455Google Scholar
  35. 35.
    Johnson AJ, Ardiani A, Sanchez-Bonilla M, Black ME (2011) Comparative analysis of enzyme and pathway engineering strategies for 5FC-mediated suicide gene therapy applications. Cancer Gene Ther 18:533–542. doi: 10.1038/cgt.2011.6 PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Madsen SJ, Sun CH, Tromberg BJ, Hirschberg H (2001) Development of a novel indwelling balloon applicator for optimizing light delivery in photodynamic therapy. Lasers Surg Med 29:406–412PubMedCrossRefGoogle Scholar
  37. 37.
    Johannesen TB, Watne K, Lote K, Norum J, Hennig R, Tverå K, Hirschberg H (1999) Intracavity fractionated balloon brachytherapy in glioblastoma. Acta Neurchiurica 141:127–133CrossRefGoogle Scholar
  38. 38.
    Eljamel MS, Goodman C, Moseley H (2008) ALA and Photofrin fluorescence-guided resection and repetitive PDT in glioblastoma multiforme: a single centre Phase III randomised controlled trial. Lasers Med Sci 23:361–367PubMedCrossRefGoogle Scholar
  39. 39.
    Hirschberg H, Zhang MJ, Gach HM, Uzal FA, Chighvinadze D, Sun CH, Peng Q, Madsen SJ (2009) Targeted delivery of bleomycin to the brain using photo-chemical internalization of Clostridium perfringens epsilon prototoxin. J Neurooncol 95:317–329. doi: 10.1007/s11060-009-9930-4 PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Madsen SJ, Gach HM, Hong SJ, Uzal FA, Peng Q, Hirschberg H (2013) Increased nanoparticle-loaded macrophage migration into the brain following PDT-induced blood-brain barrier disruption. Lasers Surg Med 45:524–532. doi: 10.1002/lsm.22172 PubMedGoogle Scholar
  41. 41.
    Cho SK, Kwon YJ (2011) Polyamine/DNA polyplexes with acid-degradable polymeric shell as structurally and functionally virus-mimicking nonviral vectors. J. Control Release. 150:287–297. doi: 10.1016/j.jconrel.2010.12.004 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Frederick Wang
    • 1
    Email author
  • Genesis Zamora
    • 1
  • Chung-Ho Sun
    • 1
  • Anthony Trinidad
    • 1
  • Changho Chun
    • 2
  • Young Jik Kwon
    • 2
    • 3
  • Kristian Berg
    • 4
  • Steen J. Madsen
    • 5
  • Henry Hirschberg
    • 1
    • 5
  1. 1.Beckman Laser Institute and Medical ClinicUniversity of California IrvineIrvineUSA
  2. 2.Department of Chemical Engineering/Material ScienceUniversity of California IrvineIrvineUSA
  3. 3.Department of Pharmaceutical SciencesUniversity of California IrvineIrvineUSA
  4. 4.Department of Radiation Biology, The Norwegian Radium HospitalOslo University HospitalOsloNorway
  5. 5.Department of Health Physics and Diagnostic SciencesUniversity of NevadaLas VegasUSA

Personalised recommendations