Cytosine deaminase/5-fluorocytosine exposure induces bystander and radiosensitization effects in hypoxic glioblastoma cells in vitro.
OBJECTIVE
Treatment of glioblastoma (GBM) is limited by therapeutic ratio; therefore, successful therapy must be specifically cytotoxic to cancer cells. Hypoxic cells are ubiquitous in GBM, and resistant to radiation and chemotherapy, and, thus, are logical targets for gene therapy. In this study, we investigated whether cytosine deaminase (CD)/5-fluorocytosine (5-FC) enzyme/prodrug treatment induced a bystander effect (BE) and/or radiosensitization in hypoxic GBM cells.


METHODS
We stably transfected cells with a gene construct consisting of the SV40 minimal promoter, nine copies of a hypoxia-responsive element, and the yeast CD gene. During hypoxia, a hypoxia-responsive element regulates expression of the CD gene and facilitates the conversion of 5-FC to 5-fluorouracil, a highly toxic antimetabolite. We used colony-forming efficiency (CFE) and immunofluorescence assays to assess for BE in co-cultures of CD-expressing clone cells and parent, pNeo- or green fluorescent protein-stably transfected GBM cells. We also investigated the radiosensitivity of CD clone cells treated with 5-FC under hypoxic conditions, and we used flow cytometry to investigate treatment-induced cell cycle changes.


RESULTS
Both a large BE and radiosensitization occurred in GBM cells under hypoxic conditions. The magnitude of the BE depended on the number of transfected cells producing CD, the functionality of the CD, the administered concentration of 5-FC, and the sensitivity of cell type to 5-fluorouracil.


CONCLUSIONS
Hypoxia-inducible CD/5-FC therapy in combination with radiation therapy shows both a pronounced BE and a radiosensitizing effect under hypoxic conditions.
Chen
Jennifer K
JK
Brain Tumor Research Center of the Department of Neurological Surgery, University of California-San Francisco, San Francisco, CA 94143, USA.
Hu
Lily J
LJ
Wang
Dongfang
D
Lamborn
Kathleen R
KR
Deen
Dennis F
DF
D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use*; D024201:Bystander Effect / Q000187:drug effects*; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D043525:Cytosine Deaminase / Q000235:genetics / Q000627:therapeutic use*; D005437:Flucytosine / Q000627:therapeutic use*; D018014:Gene Transfer Techniques; D005909:Glioblastoma / Q000503:physiopathology / Q000532:radiotherapy / Q000628:therapy*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism; D011355:Prodrugs / Q000627:therapeutic use; D011836:Radiation Tolerance / Q000187:drug effects / Q000235:genetics*
D000964:Antimetabolites, Antineoplastic; D051793:Hypoxia-Inducible Factor 1; D011355:Prodrugs; D005437:Flucytosine; D043525:Cytosine Deaminase