Mechanisms of tumor resistance to 4-hydroperoxycyclophosphamide (4-HC) were studied by using a panel of human medulloblastoma cell lines either passaged in the laboratory for resistance to 4-HC or established from tumors showing clinical resistance to cyclophosphamide. Multiple distinct mechanisms of resistance were demonstrated. Daoy (4-HCR), a line that was 6-fold more resistant than Daoy, contained elevated levels of aldehyde dehydrogenase (ALDH). Most of the difference in sensitivity between the Daoy (4-HCR) and Daoy cell lines was abolished when 4-HC was replaced with phenylketocyclophosphamide, a 4-HC analogue that cannot be detoxified by ALDH. Thus, elevated levels of ALDH appear to play a role in the resistance of Daoy (4-HCR). Several of the cell lines [D283 Med (4-HCR), D341 Med (4-HCR), Daoy (4-HCR), D458 Med] contained elevated levels of glutathione (GSH). No changes in glutathione-S-transferase activity or isozyme pattern were observed, but in two of these three lines, the elevation in GSH was accompanied by elevated levels of gamma-glutamyl transpeptidase. To confirm the role of elevated GSH content in 4-HC resistance, the sensitivity of the cell lines to 4-HC was repeated after depletion of GSH by treatment with L-buthionine-S,R-sulfoximine. In medulloblastoma cell lines without other mechanisms of resistance, a linear relationship was seen between GSH content and resistance to 4-HC. Moreover, cells with GSH content greater than 5 nmol/mg protein and no other overriding mechanism of resistance could be sensitized to 4-HC treatment with L-buthionine-S,R-sulfoximine. Finally, D283 Med (4-HCR) cells had mild elevations in both ALDH and GSH content, but were resistant to phenylketocyclophosphamide and were not significantly sensitized by L-buthionine-S,R-sulfoximine. This cell line appears to demonstrate a third mechanism of resistance to 4-HC. These results suggest that 4-HC resistance in medulloblastoma can be multifactorial.
