Application of antisense ribonucleic acid complementary to O6-methylguanine-deoxyribonucleic acid methyltransferase messenger ribonucleic acid for therapy of malignant gliomas.

OBJECTIVE: A derivative of chloroethylnitrosoureas, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), is a drug of choice for the chemotherapy of human malignant brain tumors. However, the cytocidal effect of ACNU is effectively repressed through repair of ACNU-mediated deoxyribonucleic acid lesions by O6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT). Because a variety of human tumors, including brain tumors, contain high levels of MGMT activity, we investigated the effect of antisense ribonucleic acid (RNA) complementary to MGMT messenger RNA on ACNU resistance in tumor cells. METHODS: We established a stable ACNU-resistant clone, C6AR, from the rat glioma cell line C6 exposed to a stepwise increasing concentration of ACNU. We transfected a plasmid deoxyribonucleic acid-encoding antisense MGMT RNA under the control of the human metallothionein promoter into C6AR cells and determined the effect of the antisense RNA on ACNU resistance of tumor cells by a colony-forming efficiency assay. RESULTS: C6AR cells expressed abundant MGMT messenger RNA, although the transcription level of the MGMT gene in parental C6 cells was below the lower limits of detection under the same assay conditions. ACNU resistance of C6AR cells was significantly repressed by transfected gene-dependent antisense MGMT RNA expression that resulted in decreased survival of the tumor cells. CONCLUSION: ACNU resistance resulting from the expression of MGMT in rat glioma cells is significantly overcome by the expression of antisense MGMT RNA. This result suggests that the antisense MGMT RNA system might be a useful strategy for overcoming ACNU resistance in the treatment of intractable malignant gliomas.

Early stage detection of chemotherapeutic effect on 203 GL glioma in mice as studied by P-31 NMR and flow cytometry.

The effect of chemotherapy against glioma in mouse was evaluated by 31P NMR spectroscopy and flow cytometry. We found that administration of ACNU or tegafur at a dose less than LD50 resulted in the partial suppression of the ratio of inorganic phosphate (Pi)/phosphocreatine (PCr) and phosphomonoester (PME)/creatine phosphate (PCr) after 24 or 48 hr, although these ratios are usually increased together with growth of tumors. Flow cytometric analysis of glioma in vivo showed an accumulation in cells containing tetraploid DNA by G2M block 24-48 hr after treatment. However, the change occurred at a period slightly later than that of the Pi/PCr ratio. In contrast, histological change was noted at eight days after administration. Hence, it is concluded that in vivo 31P NMR spectroscopy can detect a change in metabolic pathways in tumors as early as 24-48 hr after the administration of chemotherapeutic agents.

[Application of flow cytometry to chemotherapy of malignant brain tumor].

It has been suggested that flow cytometric analysis may offer an ability to select drugs for chemotherapy of malignant neoplasms. For this purpose perturbation of cell cycle travers which induced by several anti-cancer drugs were studied to determine the fundamental factors to evaluate the effectiveness of therapy for individual tumors. From these results, we have made a presumption that for the majority drugs studied, the perturbation of cell cycle travers will be proportional to tumor cell kill. Primary cultured cells from the human brain tumor were used to determine the effectiveness of drugs for its treatment using Factor B (the accumulated cells in SG2M phases after anti-cancer drug treatment as the percentage of cells that was previously in SG2M phases) in comparison with the results (dose-response curves) obtained by glioma cell line. The clinical application was tried using these results. A case with malignant astrocytoma had shown 20.8% for ACNU treatment, however, 85.7% for VCR treatment in maximum range of Factor B on the samples of the removed tumor at the operation (cultured cells). This patient was already treated with radiation, ACNU and other anti-cancer drugs but subsequently failed and revealed constant growth in tumor size. Thereafter patient was treated with VCR according to flow cytometric indication, there was a response, that was the first time after the desperate trials of various drugs. It was only one case, nevertheless, this result illustrates the type of studies for our plan to pursue in order to determine if flow cytometric analysis aids in the brain tumor chemotherapy by individualizing patient's treatment in near future.