EXPERIMENTAL STUDIES Quinacrine Enhances Carmustine Therapy of Experimental Rat Glioma Sandra Reyes, M.Sc., Luis A. Herrera, Ph.D., Patricia Ostrosky, Ph.D., Julio Sotelo, M.D. Neuroimmunology Unit (SR, JS), Instituto Nacional de Neurología y Neurocirugía, and the Toxicology Laboratory (LAH, PO), Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico OBJECTIVE: The high rate of mutagenesis in malignant cells has been considered to be a primary factor in the appearance of chemotherapy-resistant cell clones in glioblastomas. Quinacrine binds strongly to deoxyribonu- cleic acid, preventing mutagenesis. We investigated whether quinacrine could improve carmustine therapy in C6 cell cultures and in C6 malignant gliomas implanted subcutaneously into Wistar rats. METHODS: A potential chemopreventive effect of quinacrine on acquired resistance to carmustine therapy was studied in vitro and in vivo. Deoxyribonucleic acid damage was measured in cultured C6 cells by using the micronucleus test. Wistar rats with subcutaneously implanted C6 gliomas were treated with carmustine, quin- acrine, or carmustine plus quinacrine, using pharmacological schemes similar to those used for human patients. RESULTS: The addition of quinacrine to cultured C6 cells did not modify carmustine-induced cytotoxicity; however, the deoxyribonucleic acid damage in surviving cells was minor, as indicated by the frequency of micronucleated cells. The surviving cells continued to be susceptible to a second exposure to carmustine, in contrast to non-quinacrine-treated control cells, which developed resistance to carmustine in a subsequent exposure (P < 0.05). The rate of tumor remission was higher for glioma-bearing rats treated with quinacrine plus carmustine, compared with rats treated with carmustine alone (P < 0.01). CONCLUSION: The addition of quinacrine to carmustine therapy increases the antineoplastic effect of the carmus- tine therapy. Our results suggest that chemical inhibition of mutagenesis in malignant glial cells during chemo- therapy prevents the appearance of resistant clones. (Neurosurgery 49:969–973, 2001) Key words: Chemotherapy, Micronuclei, Mutagenesis T he therapeutic outcome of malignant gliomas is poor. Many factors may contribute to the failure of treatment; however, the emergence of cells resistant to chemother- apy is the most frequent (19). Acquired drug resistance may be caused either by genetic changes in originally sensitive tumor cells or by selective outgrowth of preexisting resistant cells (5). Some antineoplastic drugs, such as the chloroethylni- trosoureas, induce mutagenicity, which might lead to the appearance of resistant clones of neoplastic cells that were initially sensitive to the drug (7, 11, 14, 18). This seems to be consistent with clinical observations of tumors becoming un- responsive to chemotherapeutic agents that were previously effective (15). Quinacrine was an early antimalarial drug that has been almost abandoned in human therapy; however, it is widely used in experimental biology for its remarkable intracellular actions (10). Quinacrine is one of the most potent antimuta- genic agents in bacteria and eukaryotic cells, and it reduces the frequency of mutations in leukemia cells, preventing the emergence of resistant clones and thus rendering the neoplas- tic cells more susceptible to chemotherapy (2, 6, 13, 20). We investigated whether quinacrine treatment could improve the therapeutic effects of carmustine in C6 glioma cells and C6 rat gliomas. MATERIALS AND METHODS In vitro studies were performed as follows. C6 glioma cells were obtained from the American Type Culture Collection (Rockville, MD) and cultured at 37°C under sterile conditions, in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. The direct effects of quinacrine on the cytotoxicity produced by carmustine was studied by plating 600 exponentially growing C6 cells on 60-mm dishes in 5 ml 969 Neurosurgery, Vol. 49, No. 4, October 2001