Experiments investigating AGP identity, direct drug-binding capacity, and influence on STI571 activity in vitro.
(A) Representative Western blot of isolated CML-derived AGP. Briefly, following probing with a mouse antihuman α1-acid glycoprotein monoclonal antibody (clone AGP42 Sigma, Poole, United Kingdom) and a secondary antimouse alkaline phosphatase–conjugated antibody (Sigma), the blot was developed with NitroBlue tetrazolium bromochloroindolyl phosphate (NBT-BCIP). (B) Measurement of AGP drug-binding capacity by fluorescence quenching. Neither normal-derived nor CML-derived AGP (1 mg/mL) bound 1 μM STI571, as indicated by lack of quenching of the glycoprotein's fluorescence (solid lines). Peak fluorescence of the CML-derived AGP alone at λmax (335 nm) was 49.02 ± 0.35 arbitrary units (mean ± SD; n = 5). Both normal-derived and CML-derived AGP at 1 mg/mL could bind 2.5μM chlorpromazine (dotted lines; + CP) as shown by fluorescence quenching. (C) Effect of CML-derived AGP on K562 proliferation ± STI571. CML-derived AGP was tested for its ability to block the effect of 1 μM STI571 on K562 cell proliferation in vitro by a standard3H-thymidine–uptake assay. AGP isolated from the plasma of patients A and B was tested at 1 mg/mL, and from patients C and F at 2 mg/mL final concentration. Errors are displayed as 1 SD about the mean of triplicate determinations. (D) Effect of normal AGP on K562 proliferation ± STI571. Normal AGP (0.1 to 5 mg/mL) was tested for an ability to block the effect of STI571 (0.1 to 10 μM) on K562 cell proliferation in vitro. K562 cell proliferation in the absence of STI571 or AGP is indicated by the hashed line (— · —); SEM around this mean is indicated by the dotted line (-----).
