Purification and characterization of tubulin from parental and vincristine-resistant HOB1 lymphoma cells.
A multidrug-resistant lymphoma cell line resistant to 1.0 microM vincristine (designated HOB1/VCR1.0) was established. The tubulins of parental and resistant cell lines were purified by ion-exchange chromatography. Two-dimensional polyacrylamide gel electrophoresis of tubulins showed a decrease in the basic component of beta-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 x 10(6), and that for HOB1/VCR1.0 cells was 3.1 x 10(6). 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 x 10(5) and 2.0 x 10(5), 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.
Lee
W P
WP
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004351:Drug Resistance; D006801:Humans; D008223:Lymphoma / Q000378:metabolism*; D014316:Tritium; D014404:Tubulin / Q000302:isolation & purification* / Q000378:metabolism; D014750:Vincristine / Q000378:metabolism*
D014404:Tubulin; D014316:Tritium; D014750:Vincristine