P-glycoprotein is hyperphosphorylated in multidrug resistant HOB1 lymphoma cells treated with overdose of vincristine

Two proteins with M(r) values of 170 kDa and 200 kDa, respectively, were identified in HOB1 lymphoma cells resistant to 1.0 microM vincristine (designated HOB1/VCR1.0) by Western blot. Using anti-P-glycoprotein monoclonal antibody to immunoprecipitate the protein from the 32P-labeled extract of HOB1/VCR1.0 cells, the major form of the protein was in the area of 200 kDa. When the cells were cultured in 0.5 microM vincristine for 72 h, the major form shifted to the area of 170 kDa. Northern blot analysis of the mdr transcription showed the gene had been overexpressed to a maximum in the cells resistant to 0.5 microM vincristine. [3H]Vincristine uptake study showed the cells with hyperphosphorylated P-glycoprotein accumulated only half the amount of the agent after 60 min of incubation as compared with those with hypophosphorylated protein. The current study suggests hyperphosphorylated P-glycoprotein is a form by which the cells can effectively exploit the protein when it can not be induced any more.