Biochemical genetic analysis of indanocine resistance in human leukemia

Indanocine is a potent tubulin-binding drug that is cytotoxic to multidrug- resistant cancer cell lines. We demonstrated that indanocine specifically i nduces apoptosis in malignant B cells from patients with chronic lymphocyti c leukemia. To address the exact biochemical basis for indanocine toxicity, an indanocine-resistant clone was selected from mutagenized CEM human lymp hoblastoid cells. The resistant cells displayed a stable indanocine-resista nt phenotype for at least 9 months in drug-free culture. The cloned cells a re cross-resistant to colchicine and vinblastine, but not to paclitaxel, an d do not have increased expression of the multidrug-resistant p170 glycopro tein. In both parental cells and cell extracts, indanocine treatment caused tubulin depolymerization. In contrast, the tubulin in the resistant clone did not depolymerize under identical conditions. Both extract mixing and ce ll fusion experiments suggested that a stable structural change in microtub ules, rather than a soluble factor, was responsible for indanocine resistan ce. Sequence analysis of parental and resistant cells revealed a single poi nt mutation in the M40 isotype of beta -tubulin at nucleotide 1050 (G-->T, Lys(350)-->Asn) in the indanocine-resistant clone, in a region close to the putative colchicine binding site.