Cytogenetic detection of a trans-species bystander effect: induction of sister chromatid exchanges in murine 3T3 cells by ganciclovir metabolized in HSV thymidine kinase gene-transfected Chinese hamster ovary cells.
Due to the very limited transduction capacity of hitherto available vectors, the success of gene therapy of tumours by means of suicide genes has so far essentially depended on the transfer of toxic drug metabolites from transduced (metabolizing) cells to adjacent non-transduced cells via gap junctions (bystander effect). Most experimental systems for the detection of a bystander effect yield net data of cell losses and cannot differentiate between killed transduced versus killed bystander cells. Here we report on metabolic cooperation in vitro between CHO cells stably transfected with the thymidine kinase gene of herpes simplex virus type-1 (HSVtk) (metabolizing cells) and Swiss albino 3T3 cells (bystander cells). Both cell lines are readily distinguishable by single cell and colony morphology and by their chromosomal constitution. While 3T3 cells cultured alone were refractory to the antiviral drug ganciclovir (GCV), co-culture with CHO-HSVtk(+) cells led to a dramatic reduction in plating efficiency as well as to a 4-fold increase in sister chromatid exchange rates induced by very low GCV concentrations in the 3T3 bystander cells. The modulator of gap junctional cooperation, all-trans retinoic acid, caused a strong augmentation of the bystander effect, while 18alpha-glycyrrhetinic acid, an inhibitor of gap junctional communication, drastically diminished the toxicity of GCV in the bystander cells. Whereas CHO-HSVtk(+) cells showed a distinct immunoreactivity for connexin43 in the cell membranes, 3T3 cells were almost negative. The co-culture system described here allows unequivocal distinction between metabolizing and bystander cells. In this way, mechanistic aspects of the transfer of genotoxic/cytotoxic metabolites to cells, which per se are unable to form them, become accessible to investigation.
Thust
R
R
Institute of Virology and Antiviral Therapy, Medical Faculty, Friedrich Schiller University of Jena, Winzerlaer Strasse 10, D-07745 Jena, Germany. rudolf.thust@med.uni-jena.de
Tomicic
M T
MT
Gräbner
R
R
Friedrichs
C
C
Wutzler
P
P
Kaina
B
B
D016475:3T3 Cells; D000818:Animals; D024201:Bystander Effect; D016466:CHO Cells; D018031:Connexin 43 / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D020732:Cytogenetic Analysis / Q000379:methods*; D015774:Ganciclovir / Q000378:metabolism* / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods; D006034:Glycyrrhetinic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D018259:Herpesvirus 1, Human / Q000235:genetics; D051379:Mice; D012854:Sister Chromatid Exchange / Q000187:drug effects*; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
D018031:Connexin 43; C119129:18alpha-glycyrrhetinic acid; D013937:Thymidine Kinase; D006034:Glycyrrhetinic Acid; D015774:Ganciclovir