Fachbereich Veterinärmedizin



    Ganciclovir-Prodrug-Therapie von nichtkleinzelligen Bronchialkarzinomzellinien im murinen Xenotransplantationsmodell (2002)

    Haye, Sieglinde
    Berlin, 2002 — 133 Seiten
    Institut für Tierpathologie

    Robert-von-Ostertag-Str. 15
    Gebäude 12
    14163 Berlin
    +49 30 838 62450

    Abstract / Zusammenfassung

    This study investigates the therapeutic effect of ganciclovir (GCV) on human nonsmall cell lung cancer (NSCLC) cell lines which were transduced with a modified herpes simplex virus-thymidine kinase (HSV-TK30) gene in a murine xenotransplant model.The HSV-TK/GCV-system was optimized using a HSV-TK-mutante -HSV-TK30- and a vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped LXIN based rKat-vector.HSV-TK enables the transduced cells to convert the non-toxic prodrug ganciclovir into the toxic ganciclovir triphosphate (GCV-TP) form. GCV-TP inhibits the DNA polymerase and causes the death of cells.One of the most promising aspects observed in the HSV-TK/GCV system is that tumor cells that are not actually expressing the HSV-TK gene, but that are merely in close proximity to HSV-TK transduced cells, are killed after exposure to GCV. The so called "metabolic" ystander effect is extremely important for achieving effective therapeutic responses against cancer, as it is improbable to introduce a therapeutic gene into all cells of a tumour using the currently available gene transfer techniques. All entire tumour can be eliminated when only a fraction of its tumour cells express HSV-TK. The bystander effect acts as a result of metabolic cooperation ill which toxic metabolites of GCV phophorylation are passed via gap junctions between eighbouring cells. In addition, an "immunological" bystander effect exists, which is necessary for complete remission of the tumour in vivo but it is not available in immune deficient mice.This study determined the influence of the metabolic bystander effect on the ganciclovir therapy. For this, we used 100% HSV-TK30 ransduced NSCLC cells (HSV-TK) and mixed cultures that contained 25% HSV-TK30 transduced cells to 75% EGFP transduced cells (bystander). As a control only EG17P transduced cells (EGFP) were used.Stably HSV-TK30 transduced cell lines -KNS 62 and Colo 699- were established using a VSVG pseudotyped retroviral vector.The in vitro cytotoxicity assay demonstrates a significantly higher sensitivity to GCV for HSV-TK30 cells, compared to the EGFP controls. Significant growth inhibition was only observed in the KNS 62 bystander cultures, but not in the Colo 699 ystander cultures.Using immunfluorescent microscopy and western blot, it was possible to observe a definite increase in the amount of Connexin43 (Cx43) -normally forming gap junctionsin KNS 62 cells in contrast to Colo 699 cells.Using a subcutaneous mouse model 100% HSV-TK30 transduced tumours of KNS 62 and Colo 699 respectively regressed during the GCV-therapy, but tumours developed again after en days without treatment. Only the bystander tumours of the KNS 62 cells retained significant growth inhibition.Ill the orthotopic mouse model a significant reduction of mediastinal and pleurovisceral metastases was detected for both HSV-TK30 tumours (KNS 62 and Colo 699), as well as in the KNS 62 bystander tumours during GCV therapy.Ganciclovir treatment of mice with orthotopic inoculated 100% HSV-TK30 transduced KNS 62 cells and Colo 699 cells revealed a statistical significant prolonged survival for more than 80%. The mice bearing a KNS 62 bystander tumour achieved a benefit in survival for 65%. The mice bearing a Colo 699 bystander tumour had an advantage in survival for only 11 % compared to the EGFP control group.A connection between a low Cx43 expression and a minor metabolic bystander effect was observed for the Colo699 cell line, both in vitro and in vivo, espectively. KNS 62 which had a strong Cx43 expression in vitro showed an effective metabolic bystander effect in vitro and in vivo respectively.After GCV therapy subcutaneous tumours were screened for HSV-TK30 gene expression by immunoblot. 100% HSV-TK30 transduced KNS 62 tumours had weak HSV-TK30 expression, while the 100% HSV-TK30 transduced Colo 699 tumours and the bystandertumours had no detectable amount of HSV-TK30 protein. This result was explained by the decrease of HSV-TK30 gene expression.This study demonstrated that the HSV-TK/GCV system is a potent therapeutic approach for the investigated NSCLC cell lines. Also, the immunological bystander effect, which is essential for a complete tumour regression in vivo, was not functioning in immune deficient mice. This refined HSV-TK/GCV system using the mutant HSV-TK30 and tile VSV-G pseudotyped retroviral vector seems to be a promising approach for clinical trials.