MGMT- and P450 3A-inhibitors do not sensitize glioblastoma cell cultures against nitrosoureas

Aim. material and methods: Using RT-PCR and immunohistochemistry the expres sion of cytochrome P450 was evaluated in a series of 22 glioblastomas and 4 anaplastic astrocytomas (WHO grade III). Since rat liver P450 can catalyze the denitrosation of the nitrosourea compound BCNU in vitro, cell culture experiments were performed to test a possible sensitizing effect of P450 3A inhibitors (tiamulin and ketoconazole) in BCNU treatment of glial tumor ce lls. O6-benzylguanine (BG), an inhibitor of the DNA repair enzyme O6-methyl guanine-DMNA-methyltransferase (MGMT), was used in parallel experiments, si nce MGMT is discussed as a main mechanism in nitrosourea resistance.Results : RT-PCR reactions with primers designed according to the sequences for CYP 1A1 and CYP2E1 were always positive, while those for CYP1A2 and CYP2D6 were negative. The strongest PCR products were detected with CYP3A primers, but CYP3A expression was heterogeneous within the tumor samples. Antibodies to human liver CYP3A4 stained a subfraction of tumor cells (18% of the cells in glioblastomas and 14% in grade III astrocytomas) and to some extend neur ons in normal brain areas, while astrocytes were negative. For cell culture experiments with P450 3A and MGMT inhibitors, early passages of 3 glioblas tomas, a late passage of an immortalized cell line derived from a reoccurri ng glioblastoma, and the human glioblastoma line LN405 were used. The sensi tivity of the tumor cells for both nitrosourea compounds was very low, when low concentrations were applied (comparable to the achievable blood concen trations in glioma patients). Strong effects did only occur when the concen trations were raised 9-fold or 27-fold.Conclusion: In no case could a signi ficant sensitizing effect of P450 3A- and MGMT inhibitors be demonstrated.