gms | German Medical Science

Objective: The Hedgehog signalling pathway is important for the development of the central nervous system. On the other hand, aberrant induction is observed in different tumors. Immunofluorescence and RT-PCR confirmed that in some gliomas Gli1, a transcription factor activated by signalling, is present. In general, the hedgehog pathway is initiated by binding of extracellular ligands to the transmembrane receptor Patched and leads finally to the activation of the transcription factors Gli1, Gli2 and Gli3. Whereas Gli1 acts as an activator, Gli2 appears to be an activator but retains some repressor activities and Gli3 is believed to act only as an inhibitor. Therefore, the determination of hedgehog activity at the level of transcription requires additional experiments measuring gene activation.

Methods: Cells isolated from tumors of patients with glioblastoma (WHO Grade IV) and cells from two different glioma cell lines were transfected with reporter genes. The reporter genes carried the luciferase from Gaussia princeps under the control of promoters conjugated to Gli binding sites. The activity of the reporter genes was analysed in the absence and presence of the hedgehog signalling inhibitor cyclopamine. In addition the effect of cyclopamine on cellular metabolism was studied.

Results: The analysis revealed that the two cell lines and cells from 6 glioblastomas exhibited enhanced reporter gene expression. In three cultures a repression of activity was detected and four cultures did neither show activation nor repression. Enhanced luciferase activity in cells from the line T98G and in cells from three primary cultures was not influenced by the hedgehog inhibitor cyclopamine, whereas one cell line significantly responded to its presence. Interestingly, ATP production was suppressed by cyclopamine in cells from the line T98G and also in cells from one primary culture that responded to the inhibitor.

Conclusions: With the newly developed reporter genes transcriptional enhancement as well as silencing due to hedgehog signalling can be detected. In fact, for the first time the present study demonstrates a repression of transcription in glioblastomas due to hedgehog signalling. The test system established in the present work may be a useful diagnostic tool to evaluate the prospects of a therapy targeting hedgehog signalling for the treatment of malignant glioma. In addition, it is demonstrated that cyclopamine may be considered as a therapeutic agent even in the absence of an influence on Gli activity.