Purification and Characterization of Tubulin from Parental and Vincristine-Resistant HOB1 Lymphoma Cells

A multidrug-resistant lymphoma cell line resistant to 1.0 μM vincristine (designated HOB1/VCR1.0) was established. The tubulins of parental and resistant cell Lines mere purified by ion-exchange chromatography. Two-dimensional polyacrylamide gel electrophoresis of tubulins showed a decrease in the basic component of β-tubulin in the HOB1/VCR1.0 cell line; native isoelectric focusing of tubulins showed decreased expression of two more basic tubulin dimers in the same cell line. The [3H]vincristine-tubulin binding studies were performed by filtration and HPLC and displayed the tubulin of HOB1/VCR1.0 cells having a weaker binding affinity to vincristine than those of parental HOB1 and HOB1/VCR0.5 cells. The binding constant Ka of purified tubulin to vincristine, calculated from the slope of the Scatchard curve, for parental HOB1 cells was 5.6 × 106, and that for HOB1/VCR1.0 cells was 3.1 × 106. The Scatchard kinetics was also used to determine the binding ability of the purified tubulins to [3H]colcemid: the Kas for parental and HOB1/VCR1.0 cells were 3.9 × 105 and 2.0 × 105, respectively. The current study suggests that high-level resistant cells, HOB1/VCR1.0, tend to express fewer tubulin isoforms of stronger binding affinities to antimitotic agents; that is, they preserve weak drug-binding forms rather than produce additional species. This may be a mechanism for the cells to protect themselves from drug injury when the P-glycoprotein cannot efficiently pump out the agent of high concentration within the cells.