Multiple microtubule alterations are associated with Vinca alkaloid resistance in human leukemia cells

Vinea alkaloids are used extensively in the treatment of childhood acute ly mphoblastic leukemia (ALL) and despite their usefulness, drug resistance re mains a serious clinical problem. Vinca alkaloids bind to the beta -tubulin subunit of the alpha/beta -tubulin heterodimer and inhibit polymerization of microtubules. Recent studies have implicated altered beta -tubulin isoty pe expression and mutations in resistance to microtubule-stabilizing agents . Microtubule-associated protein (MAP) MAP4 binds to and stabilizes microtu bules, and increased expression is associated with decreased sensitivity to microtubule-depolymerizing agents. To address the significance of beta -tu bulin and MAP4 alterations in childhood ALL, two CCRF-CEM-derived Vinca alk aloid resistant cell lines, VCR R (vincristine) and VLB100 (vinblastine), w ere examined. Decreased expression of class III beta -tubulin was detected in both VCR R and VLB100 cells. VCR R cells and to a lesser extent VLB100 c ells expressed increased levels of MAP4 protein. Increased microtubule stab ility was observed in these VCR R cells as identified by the high levels of polymerized tubulin (45.6 +/- 2.6%; P < 0.005) compared with CEM and VLB10 0 cells (24.7 +/- 3.3% and 24.7 +/- 2.5 %, respectively). Expression was as sociated with a single MAP4 isoform in the polymerized microtubule fraction in CEM and VCR cells. In contrast, VLB100 cells expressed a lower molecula r weight isoform, in the polymerized fraction. Two-dimensional-PAGE and imm unoblotting revealed marked posttranslational changes in class I beta -tubu lin in VCR R cells not evident in CEM cells. Sequencing of the ig-tubulin ( HM40) gene identified a point mutation in VCR R cells in nucleotide 843 (CT C --> ATC; Leu(240)--> Ile) that was not present in CEM or VLB100 cells. Th is mutation resides in a region of beta -tubulin that lies in close proximi ty to the alpha/beta tubulin interface. Multiple alterations related to nor mal microtubule function were identified in ALL cells selected for resistan ce to Vinca alkaloids, and these alterations may provide important insight into mechanisms mediating resistance to Vinca alkaloids.