This project has continued to focus upon potential mechanisms underlying the selective brain tumor cell line cytotoxicity of quaternized ellipticines. Previous studies have demonstrated that sensitivity of brain tumor cell lines correlates (p<0.0001) with peak cellular concentration of radioactivity derived from MMEA which are achieved 10-15 hrs following initiation of drug exposure. In an attempt to determine the mechanism responsible for this difference in MMEA accumulation between sensitive and resistant cell lines, we have isolated plasma membrane from the 9-methoxy-N2-methylellipticinium (MMEA) sensitive U-251 glioblastoma cells and the MMEA-resistant XF-498 gliosarcoma. Plasma membrane from each cell line was extracted at 4 degrees C in buffer containing 6% of the non-ionic detergent n-octylglucoside for 60 min, centrifuged at 105,000xg for 60 min and the solubilized proteins in the supernatant separated on 10% non-denaturing polyacrylamide gels. The gels were silver stained and 2 bands of approximately 66 and 53 kDa were identified as being overexpressed in the U-251 glioblastoma relative to the XF-498 gliosarcoma. The molecular weights of these 2 proteins are equivalent to the known molecular weights of the dopamine transporter and variants of cytochrome P-450. Western blot analysis of plasma membrane from U-251 and XF-498 using an anti-rat IIB1/IIB2 cytochrome P-450 antibody suggests that cytochrome P-450 is present in plasma membrane from both cell lines and that it is overexpressed in U-251 relative to XF-498. Currently, in collaboration with LDDRD chemists, we are attempting to synthesize the 10-azido analog of MMEA for use as a photoaffinity label to identify the proteins responsible for MMEA accumulation.
