Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene.
The ability of herpes simplex virus type 1 thymidine kinase (HSV-tk)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-tk-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs via the transfer of phosphorylated GCV, the mechanism(s) of this bystander effect and the importance of gap junctions for the effect of prodrug/suicide gene therapy in primary human glioblastoma cells remains elusive. Surgical biopsies of malignant gliomas were used to establish explant primary cultures. Proliferating tumor cells were characterized immunohistochemically and found to express glial tumor markers including nestin, vimentin, glial fibrillary acidic protein (GFAP), S-100, and gap junction protein connexin 43 (Cx43). Western blot analysis revealed the presence of phosphorylated isoforms of Cx43 and Calcein/DiI fluorescent dye transfer showed evidence of efficient gap junction communication (GJC). In order to study the effect(s) of prodrug/suicide gene therapy in these cultures, human glioblastoma cell cultures were transfected with the HSVtk gene for transient or stable expression. Ganciclovir treatment of these cultures led to >90% of cells dead within 1 week. Eradication of cells could be inhibited by the addition of alpha-glycyrrhetinic acid (AGA), a GJC inhibitor. In parallel experiments, AGA decreased the immunodetection of phosphorylated Cx43 as analyzed by Western blot and inhibited fluorescent dye transfer. In conclusion, these observations are consistent with GJC as the mediator of the bystander effect in primary cultures of human glioblastoma cells by the transfer of phosphorylated GCV from HSVtk gene transfected cells to untransfected ones.
Asklund
Thomas
T
Department of Clinical Neuroscience, Section of Neurosurgery, Karolinska Institutet, S-171 76, Stockholm, Sweden
Appelskog
Ioulia B
IB
Ammerpohl
Ole
O
Langmoen
Iver A
IA
Dilber
M Sirac
MS
Aints
Alar
A
Ekström
Tomas J
TJ
Almqvist
Per M
PM
D014408:Biomarkers, Tumor; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects* / Q000235:genetics; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D018031:Connexin 43 / Q000378:metabolism; D003603:Cytotoxins / Q000235:genetics / Q000494:pharmacology* / Q000627:therapeutic use; D015774:Ganciclovir / Q000494:pharmacology* / Q000627:therapeutic use; D017629:Gap Junctions / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D005910:Glioma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D006034:Glycyrrhetinic Acid / Q000494:pharmacology / Q000627:therapeutic use; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D013937:Thymidine Kinase / Q000235:genetics / Q000494:pharmacology* / Q000627:therapeutic use; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics / Q000494:pharmacology* / Q000627:therapeutic use
D014408:Biomarkers, Tumor; D018031:Connexin 43; D003603:Cytotoxins; D014764:Viral Proteins; C099241:thymidine kinase, Canid herpesvirus 1; D013937:Thymidine Kinase; D006034:Glycyrrhetinic Acid; D015774:Ganciclovir